Biología de la Diferenciación Celular

Tema 2

Mecanismos celulares y moleculares de

la fecundación animal. Reproducción
asistida. Clonación
 Tema 2. Biología de la Diferenciación Celular

1. Introducción

2. Mecanismos celulares y moleculares de la fecundación

animal

3. Técnicas de reproducción asistida

4. Clonación
1. Introducción
 Cromosomas en la meiosis y en la fecundación

Figure 3.2. Chromosomes at meiosis and fertilization Two chromosome pairs of a hypothetical organism are illustrated. (Cooper y Hausman, 2004).
Primeros estadíos del desarrollo embrionario humano
El ciclo menstrual

Figure 19.30. The human menstrual cycle. The

coordination of (B) ovarian and (D) uterine
cycles is controlled by (A) the pituitary and (C)
the ovarian hormones. During the follicular
phase, the egg matures within the follicle, and
the uterine lining is prepared to receive the
blastocyst. The mature egg is released around
day 14. If a blastocyst does not implant into the
uterus, the uterine wall begins to break down,
leading to menstruation. (Gilbert, 2003).
Gametogénesis

1. Estadíos del desarrollo y características de los oocitos

2. Estadíos del desarrollo y características de los espermatozoides

1. Estadíos del desarrollo y características de
los oocitos
Células germinales primordiales

Figure 21-17. Migration of mammalian primordial germ cells (PGCs). Alberts et al., 2008.
 Estadíos de la oogénesis

Figure 21-23. The stages of oogenesis. Oogonia develop from primordial

germ cells that migrate into the developing gonad early in embryogenesis.
After a number of mitotic divisions, oogonia begin meiotic division I, after
which they are called primary oocytes. In mammals, primary oocytes are
formed very early (between 3 and 8 months of gestation in the human
embryo) and remain arrested in prophase of meiotic division I until the
female becomes sexually mature. At this point, a small number periodically
mature under the influence of hormones, completing meiotic division I to
become secondary oocytes, which eventually undergo meiotic division II to
become mature eggs (ova). The stage at which the egg or oocyte is released
from the ovary and is fertilized varies from species to species. In most
vertebrates, oocyte maturation is arrested at metaphase of meiosis II and the
secondary oocyte completes meiosis II only after fertilization. All of the polar
bodies eventually degenerate. In most animals, the developing oocyte is
surrounded by specialized accessory cells that help to isolate and nourish it
(not shown). (Alberts et al., 2008).
 Oocitos primarios en desarrollo

Figure 21-25. The stages of oogenesis. Oogonia develop from primordial germ cells that migrate into the developing gonad early in embryogenesis. After a number of
mitotic divisions, oogonia begin meiotic division I, after which they are called primary oocytes. In mammals, primary oocytes are formed very early (between 3 and 8
months of gestation in the human embryo) and remain arrested in prophase of meiotic division I until the female becomes sexually mature. At this point, a small number
periodically mature under the influence of hormones, completing meiotic division I to become secondary oocytes, which eventually undergo meiotic division II to
become mature eggs (ova). The stage at which the egg or oocyte is released from the ovary and is fertilized varies from species to species. In most vertebrates, oocyte
maturation is arrested at metaphase of meiosis II and the secondary oocyte completes meiosis II only after fertilization. All of the polar bodies eventually degenerate. In
most animals, the developing oocyte is surrounded by specialized accessory cells that help to isolate and nourish it (not shown). (Alberts et al., 2008).
 Oogénesis

Figure 19.29. The ovarian follicle of mammals. (A) Maturation of the ovarian follicle. When mature, it is often called a Graafian
follicle. (B) Scanning electron micrograph of a mature follicle in the rat. The oocyte (center) is surrounded by the smaller granulosa
cells that will make up the cumulus. (Gilbert, 2003).
 Ovulación

Figure 19.31. Ovulation in the rabbit. The ovary of a living, anesthetized rabbit was exposed and observed.
When the follicle started to ovulate, the ovary was removed, fixed, and stained. (Gilbert, 2003).
2. Estadíos del desarrollo y características de
los espermatozoides
 Estadíos de la espermatogénesis

Figure 21-
21-30. The stages of spermatogenesis.
spermatogenesis. Spermatogonia develop from
primordial germ cells that migrate into the testis early in embryogenesis.
embryogenesis. When the
animal becomes sexually mature,
mature, the spermatogonia begin to proliferate rapidly,
rapidly,
generating some progeny that retain the capacity to continue dividing indefinitely
(as stem-
stem-cell spermatogonia)
spermatogonia) and other progeny (maturing spermatogonia)
spermatogonia) that
will,
will, after a limited number of further mitotic division cycles,
cycles, embark on meiosis
to become primary spermatocytes.
spermatocytes. The primary spermatocytes continue through
meiotic division I to become secondary spermatocytes.
spermatocytes. After they complete
meiotic division II, the secondary spermatocytes produce haploid spermatids,
spermatids,
which differentiate into mature sperm (spermatozoa).
spermatozoa). Spermatogenesis differs
from oogenesis in several ways.
ways. (1) New cells enter meiosis continually from the
time of puberty.
puberty. (2) Each cell that begins meiosis gives rise to four mature gametes
rather than one.
one. (3) Mature sperm form by an elaborate process of cell
differentiation that begins after meiosis is complete. (4) About twice as many cell
divisions occur in the production of a sperm as in the production of an egg;
egg; in a
mouse,
mouse, for example,
example, it is estimated that on average about 56 divisions occur from
zygote to mature sperm,
sperm, and about 27 divisions occur from zygote to mature egg.
egg.
(Alberts et al., 2008).
 Estructura de los tubos seminíferos de un mamífero

Figure 21-
21-29. Highly simplified drawing of a cross section of a seminiferous tubule in a mammalian testis. testis. (A) All of the stages of spermatogenesis shown take
place while the developing gametes are in intimate association with Sertoli cells.
cells. These large cells extend from the basal lamina to the lumen of the seminiferous tubule;
tubule;
they are required for the survival of the germ cells and are analogous to follicle cells in the ovary (see Figure 20-
20-18).
18). Spermatogenesis also depends on testosterone
secreted by Leydig cells,
cells, located between the seminiferous tubules.
tubules. (B) Some of these cells are self-
self-renewing stem-
stem-cell spermatogonia,
spermatogonia, whereas others are maturing
spermatogonia;
spermatogonia; after a number of mitotic divisions,
divisions, the maturing spermatogonia stop dividing by mitosis and enter meiosis to become primary spermatocytes.
spermatocytes. Eventually,
Eventually,
sperm are released into the lumen. In man,
man, it takes about 24 days for a spermatocyte to complete meiosis to become a spermatid and another 5 weeks for a spermatid to
develop into a sperm.
sperm. Sperm undergo further maturation and become motile in the epididymis;
epididymis; only then are they fully mature sperm.
sperm. (Alberts
(Alberts et al., 2008).
 El espermatozoide humano

Figure 21-27. A human sperm. It is shown in
longitudinal section. (Alberts et al., 2008).
Figure 21-28. Drawing of the midpiece of a mammalian sperm as seen in cross section in an
electron microscope. The core of the flagellum is composed of an axoneme surrounded by nine
dense fibers. The axoneme consists of two singlet microtubules surrounded by nine microtubule
doublets. The mitochondrion (shown in green) is well placed for providing the ATP required for
flagellar movement; its unusual spiral structure (see Figure 20-25) results from the fusion of
individual mitochondria during spermatid differentiation. (Alberts et al., 2008).
 El aparato motor del espermatozoide

Figure 7.3. The motile apparatus of the sperm. (A) Cross section of the flagellum of a
mammalian spermatozoon, showing the central axoneme and the external fibers. (B)
Interpretive diagram of the axoneme, showing the "9 + 2" arrangement of the
microtubules and other flagellar components. The schematic diagram shows the
association of tubulin protofilaments into a microtubule doublet. The first ("A") portion
of the doublet is a normal microtubule comprising 13 protofilaments. The second ("B")
portion of the doublet contains only 11 (occasionally 10) protofilaments. The dynein
arms contain the ATPases that provide the energy for flagellar movement. (C) A three-
dimensional model of an "A" microtubule. The a- and b-tubulin subunits are similar but
not identical, and the microtubule can change size by polymerizing or depolymerizing
tubulin subunits at either end. (Gilbert, 2003).
El acrosoma: un tipo de lisosoma especializado

Vesí
Vesícula
Cabeza

acrosó
acrosómica
Núcleo

Porción media

Mitocondria

Membrana plasmática
Cola

Flagelo

10 μm

x 3.000

Figure 21-27. A human sperm. It is shown in

longitudinal section. (Alberts et al., 2008).
2. Mecanismos celulares y moleculares
de la fecundación animal
 Espermatozoide de mamífero en el tracto reproductivo
de la hembra

Figure 7.13. Mammalian sperm in

the female reproductive tract. Bull
sperm adhering to the membranes of
the oviduct epithelial cells prior to
entering the ampulla. (Gilbert,
2003).
 Oocitos de hámster antes de la fecundación

Figure 7.7. Hamster eggs immediately before fertilization. (A) The hamster egg, or ovum, is encased in the zona
pellucida. This, in turn, is surrounded by the cells of the cumulus. A polar body cell, produced during meiosis, is also
visible within the zona pellucida. (B) At lower magnification, a mouse oocyte is shown surrounded by the cumulus.
Colloidal carbon particles (India ink) are excluded by the hyaluronidate matrix. (Gilbert, 2003).
 La zona pelúcida

Figure 21-22. The zona pellucida. (A) Scanning electron micrograph of a hamster egg, showing the zona pellucida. (B) A scanning
electron micrograph of a similar egg in which the zona (to which many sperm are attached) has been peeled back to reveal the underlying
plasma membrane, which contains numerous microvilli. The zona is made entirely by the developing oocyte. (Alberts et al., 2008).
 Fecundación

Figure 21-32. Scanning electron micrograph of a

human sperm contacting a hamster egg. The
zona pellucida of the egg has been removed,
exposing the plasma membrane, which contains
numerous microvilli. The ability of an individual's
sperm to penetrate hamster eggs is used as an assay
of male fertility; penetration of more than 10 25%
of the eggs is considered to be normal. (Alberts et
al., 2008).
Fecundación
 Entrada de un espermatozoide en el oocito de hámster

Figure 7.20. Entry of sperm into golden hamster egg. (A) Scanning electron micrograph of
sperm fusing with egg. The "bald" spot (without microvilli) is where the polar body has
budded off. (B) Close-up of sperm-zona binding. (C) Transmission electron micrograph
showing the sperm head passing through the zona. (D) Transmission electron micrograph of
the hamster sperm fusing parallel to the egg plasma membrane. (E) Diagram of the fusion
of the sperm acrosome and plasma membranes with the egg microvilli. (Gilbert, 2003).
Desarrollo embrionario humano
Desarrollo embrionario humano
Mecanismos implicados en el reconocimiento y
fusión de los gametos. Activación del
metabolismo del cigoto
Estructura de la zona pelúcida
 Expresión de ZP3 en el oocito de ratón

Figure 19.25. Expression of the ZP3 gene in the developing mouse oocyte. (A) Northern blot of ZP3 mRNA accumulation in embryonic
mouse tissues. A radioactive probe to the ZP3 message found it expressed only in the ovary, and specifically in the oocytes. (B-C) When
the luciferinase reporter gene is placed onto the ZP3 promoter and inserted into the mouse genome, the luciferinase message is seen only
in the developing oocytes of the ovary. C is a higher magnification of a section of B, showing two of the ovarian follicles containing
maturing oocytes. (Gilbert, 2003).
 La reacción
acrosómica

Figure 21-33. The acrosome

reaction that occurs when a
mammalian sperm fertilizes an
egg. In mice, a single
glycoprotein in the zona
pellucida, ZP3, is thought to be
responsible for both binding the
sperm and inducing the acrosome
reaction. Note that a mammalian
sperm interacts tangentially with
the egg plasma membrane so that
fusion occurs at the equator,
rather than at the tip, of the sperm
head. In mice, the zona pellucida
is about 6 mm thick, and sperm
cross it at a rate of about 1
mm/min. (Alberts et al., 2008).
 Incremento de Ca2+ en el oocito en la fertilización

Figure 15-40. Fertilization of an egg by a sperm triggering an increase in cytosolic Ca2+. This starfish egg was injected with a
Ca2+-sensitive fluorescent dye before it was fertilized. A wave of cytosolic Ca2+(red), released from the endoplasmic reticulum, is seen
to sweep across the egg from the site of sperm entry (arrow). This Ca2+ wave provokes a change in the egg cell surface, preventing the
entry of other sperm, and it also initiates embryonic development (discussed in Chapter 20). (Alberts et al., 2008).
 La reacción
cortical del oocito
Bloqueo secundario a la
poliespermia

Figure 21-34. How the cortical reaction in a

mouse egg is thought to prevent additional
sperm from entering the egg. The released
contents of the cortical granules both remove
carbohydrate from ZP3 so it no longer can bind
to the sperm plasma membrane and partly
cleave ZP2, hardening the zona pellucida.
Together these changes provide a block to
polyspermy. (Alberts et al., 2008).
 Estructura de la
fertilina

Figure 20-33. The fertilin protein in the sperm plasma

membrane. The a and b subunits, which are both
glycosylated (not shown), are noncovalently associated.
Both subunits belong to the ADAM family of proteins,
which includes proteins thought to function in either cell
adhesion or the proteolytic processing of other
transmembrane proteins (such as Notch, which is
discussed in Chapter 15). The proteolytic domain that is
normally present at the amino terminus of these proteins
is removed from the fertilin protein during sperm
maturation. (Alberts et al., 2002).
Óvulo fecundado con dos pronúcleos
 Aproximación de los pronúcleos del oocito y del
espermatozoide tras la fertilización

Figure 21-35. The coming together of the sperm and egg pronuclei after mammalian fertilization. The pronuclei migrate toward the
center of the egg. When they come together, their nuclear envelopes interdigitate. The centrosome replicates, the nuclear envelopes break
down, and the chromosomes of both gametes are eventually integrated into a single mitotic spindle, which mediates the first cleavage
division of the zygote. (Alberts et al., 2008).
 Fertilización y terminación de la meiosis

Figure 14.42. Fertilization and completion of meiosis (A) Fertilization induces the transition from metaphase II to anaphase II, leading to completion of oocyte
meiosis and emission of a second polar body (which usually degenerates). The sperm nucleus decondenses, so the fertilized egg (zygote) contains two haploid nuclei
(male and female pronuclei). In mammals, the pronuclei replicate DNA as they migrate toward each other. They then initiate mitosis, with male and female
chromosomes aligning on a common spindle. Completion of mitosis and cytokinesis thus gives rise to a two-cell embryo, with each cell containing a diploid genome.
(Cooper y Hausman, 2008).
 Fertilización
humana

Figure 21-36. Immunofluorescence

micrographs of human sperm and egg
pronuclei coming together after in vitro
fertilization. Spindle microtubules are
stained in green with anti-tubulin antibodies,
and DNA is labeled in blue with a DNA
stain. (A) A meiotic spindle in a mature,
unfertilized oocyte. (B) This fertilized egg is
extruding its second polar body and is shown
about 5 hours after fusion with a sperm. The
sperm head (left) has nucleated an array of
microtubules. (C) The two pronuclei have
come together. (D) By 16 hours after fusion
with a sperm, the centrosome that entered the
egg with the sperm has duplicated, and the
daughter centrosomes have organized a
bipolar mitotic spindle. The chromosomes of
both pronuclei are aligned at the metaphase
plate of the spindle. As indicated by the
arrows in (C) and (D), the sperm tail is
associated with one of the centrosomes.
(Alberts et al., 2008).
3. Reproducción Asistida
Número de células germinales en el ovario humano en
relación a la edad

Figure 19.21. Changes in the number of germ cells in the human ovary over the life span. (Gilbert, 2003).
 Conceptos

Esterilidad
Parejas que, siendo la mujer mayor de 30 años, no han
conseguido un embarazo tras un año de relaciones
sexuales sin contracepción

Infertilidad
Parejas que consiguen una concepción pero el embarazo
se malogra y se produce una pérdida fetal
Figure 21.3. The fates of 20 hypothetical human eggs in the United States and western Europe. Under normal conditions,
only 6.2 eggs of the original 20 would be expected to develop successfully to term. (After Volpe 1987.) (Gilbert, 2003).
Edad de la madre y tasa de abortos

Maternal age Loss rate

< 30 5%
30 - 34 7%
35 - 39 15 %
40 - 41 25 %
42 - 43 35 %
44 - 46 50 %

Miscarriage rates for women with a history of infertility tend to be higher than for fertile women.
Most (if not all) of the increased risk for miscarriage in "older" women is due to the increase in
chromosomal abnormalities (karyotype) in their eggs. This is also part of the reason for the
decline in overall fertility with aging.
Riesgo de anomalías genéticas en el recién nacido en
función de la edad de la madre

Female Risk of a live birth with any chromosomal

age abnormality

25 1 / 476

30 1 / 385

35 1 / 164

40 1 / 51

45 1 / 15
Recién nacidos vivos en función del número de oocitos
extraídos y edad de la madre
Morfología del útero y trompas de Falopio

Histerosalpingografía

Normal hysterosalpingogram
A smooth triangular uterine cavity and spill from the ends of both tubes
Morfología del útero, trompas de Falopio y ovarios
Laparoscopia

Laparoscopic view of a normal pelvis

Morfología del útero, trompas de Falopio y ovarios
Laparoscopia

A normal left tube at laparoscopy for infertility. Course of tube is marked by "T"s
Normal fimbriated end of tube is shown at "F"
Left ovary is the white structure between the uterus and tube
 El ciclo menstrual

Figure 19.30. The human menstrual cycle. The

coordination of (B) ovarian and (D) uterine
cycles is controlled by (A) the pituitary and (C)
the ovarian hormones. During the follicular
phase, the egg matures within the follicle, and
the uterine lining is prepared to receive the
blastocyst. The mature egg is released around
day 14. If a blastocyst does not implant into the
uterus, the uterine wall begins to break down,
leading to menstruation. (Gilbert, 2003).
 Inseminación artificial

Fases de la inseminación artificial

1. Estimulación del ovario con substancias inductoras de la ovulación:

desarrollo de varios óvulos

2. Preparación del semen: selección y concentración de los

espermatozoides móviles

2. Procesamiento de las muestras mediante técnicas de capacitación o

preparación seminal

3. Inseminación durante dos días seguidos después de haber inducido

la ovulación
Inseminación artificial
 Fases de la fecundación in vitro (FIV)

1. Estimulación hormonal del ovario

2. Extracción de oocitos
3. Inseminación de los oocitos obtenidos
Inseminación clásica
Inyección intracitoplasmática de espermatozoides (ICSI)
4. Cultivo in vitro hasta embrión en diferentes estadíos de desarrollo
Cultivo durante tres días en el laboratorio
Co-cultivo embrionario durante 6 días hasta el estadío de blastocisto
- Células de la trompa de Falopio
- Células del endometrio
5. Transferencia embrionaria
En la cavidad uterina por vía transcervical
En las trompas de Falopio
6. Congelación y descongelación de embriones en su caso
Fecundación in vitro (FIV)
 Fecundación in vitro convencional

Requisitos
Función ovárica activa
Madurez ovocitaria
Recuperación espermática de una cantidad mínima de
espermatozoides con buena movilidad (100.000/ml)

Indicaciones principales
Factor masculino moderado
Aglutinación espermática grave
Factor tubárico
Infertilidad causada por alteración de las
trompas de Falopio

Hydrosalpinx, fallopian tube that is blocked and dilated with fluid

This is evidence of previous pelvic inflammatory disease - PID
Volumen del ovario y número de folículos en un ciclo
menstrual normal
Normal ovarian volume and "normal" antral follicle counts

Ultrasound image of an ovary at the beginning of a menstrual cycle. No medications are being given.
The ovary is outlined in blue. There are 9 antral follicles visible - marked with red spots
The ovary has normal volume (cursors measuring ovary = 30 by 17.8mm)
This woman had regular periods and a normal response to injectable FSH
 Infertilidad: Ovario poliquístico
High ovarian volume and high antral follicle counts

Ultrasound image of an ovary at the beginning of a menstrual cycle. No medications are being given.
The ovary is outlined in blue. There are numerous antral follicles visible - marked with red spots
16 are seen in this image, this ovary had a total of 35 antrals (only 1 plane is shown above)
This is a polycystic ovary, with a higher than average antral count and volume (ovary = 37 by 19.5mm)
This woman had very irregular periods and was a "high responder" to injectable FSH medication
 Infertilidad: Ovario poliquístico

For comparison, an ovary with a mature

follicle at midcycle in a woman that ovulates
This follicle is 20mm diameter
Blue circle is around the ovary

Ultrasound image of a polycystic ovary

Note many immature small follicles (black
circles) around periphery of ovary Laparoscopic view of a typical,
Blue circle is around the ovary enlarged, polycystic ovary
 Fases de la fecundación in vitro (FIV)

1. Estimulación hormonal del ovario

2. Extracción de ovocitos
3. Inseminación de los ovocitos obtenidos
Inseminación clásica
Inyección intracitoplasmática de espermatozoides (ICSI)
4. Cultivo in vitro hasta embrión en diferentes estadíos de desarrollo
Cultivo durante tres días en el laboratorio
Co-cultivo embrionario durante 6 días hasta el estadío de blastocisto
- Células de la trompa de Falopio
- Células del endometrio
5. Transferencia embrionaria
En la cavidad uterina por vía transcervical
En las trompas de Falopio
6. Congelación y descongelación de embriones en su caso
 Estimulación hormonal del ovario
Ultrasound of multiple follicles in a stimulated ovary

What is controlled ovarian hyperstimulation?

It involves the use of some of the same medications used for induction of ovulation in women
that have anovulation. These medications are used to stimulate development of multiple mature
follicles and eggs in order to increase pregnancy rates with various infertility treatments.
 Fases de la fecundación in vitro (FIV)

1. Estimulación hormonal del ovario

2. Extracción de ovocitos
3. Inseminación de los ovocitos obtenidos
Inseminación clásica
Inyección intracitoplasmática de espermatozoides (ICSI)
4. Cultivo in vitro hasta embrión en diferentes estadíos de desarrollo
Cultivo durante tres días en el laboratorio
Co-cultivo embrionario durante 6 días hasta el estadío de blastocisto
- Células de la trompa de Falopio
- Células del endometrio
5. Transferencia embrionaria
En la cavidad uterina por vía transcervical
En las trompas de Falopio
6. Congelación y descongelación de embriones en su caso
 Obtención de los oocitos para FIV
Ultrasound of multiple follicles (3 black circles) in a
stimulated ovary
The egg aspiration procedure in progress - an egg is being
aspirated from a follicle
The needle is the bright white structure (right side) above the
3rd white dot from bottom
Ovary outlined in blue, top of vagina in red
 Fases de la fecundación in vitro (FIV)

1. Estimulación del ovario con hormonas

2. Extracción de ovocitos

3. Inseminación de los ovocitos obtenidos

Inseminación clásica
Inyección intracitoplasmática de espermatozoides (ICSI)
4. Cultivo in vitro hasta embrión en diferentes estadíos de desarrollo
Cultivo durante tres días en el laboratorio
Co-cultivo embrionario durante 6 días hasta el estadío de blastocisto
- Células de la trompa de Falopio
- Células del endometrio
5. Transferencia embrionaria
En la cavidad uterina por vía transcervical
En las trompas de Falopio
6. Congelación y descongelación de embriones en su caso
 Variaciones de FIV

1. Inyección intracitoplasmática de espermatozoide

(ICSI, intracytoplasmatic sperm injection)

2. Transferencia intratubárica de gameto

(GIFT, gamete intrafallopian transfer)

3. Transferencia intratubárica de cigoto o transferencia

embrionaria tubárica
(ZIFT, zigote intrafallopian transfer)
Inyección intracitoplásmica de espermatozoides
(ICSI)

- Es necesario un solo espermatozoide vivo para cada oocito

- Puede realizarse con muestras de semen de baja calidad

- Obtención de espermatozoides directamente del testículo o epidídimo

- Biopsia testicular
Inyección intracitoplasmática de espermatozoides
(ICSI)
Inyección intracitoplásmica de espermatozoide (ICSI)

Requisitos
Función ovárica activa
Madurez oocitaria
Existencia de, al menos, tantos espermatozoides
móviles como oocitos se hayan obtenido

Indicaciones principales
Factor masculino severo
Alteración de membrana ovocitaria
Fallo de fecundación en FIV convencional
 ICSI

Inyección de un espermatozoide dentro de un óvulo

 Procedimiento de ICSI

About to inject the egg with a sperm. Holding pipette on left

ICSI needle on right. Sperm head visible in needle at far right,
just below X. Polar body of egg at 7 o'clock Needle is advanced to the left
Shell of embryo has already been penetrated
by needle
Membrane of egg (oolemma) is stretching
and is about to break
Sperm head is visible at tip of needle

ICSI needle has penetrated the egg membrane

A single sperm is being injected into the
cytoplasm of the egg
 Fases de la fecundación in vitro (FIV)
1. Estimulación del ovario con hormonas
2. Extracción de ovocitos
3. Inseminación de los ovocitos obtenidos
Inseminación clásica
Inyección intracitoplasmática de espermatozoides (ICSI)
4. Cultivo in vitro hasta embrión en diferentes estadíos de desarrollo
Cultivo durante tres días en el laboratorio
Co-cultivo embrionario durante 6 días hasta el estadío de blastocisto
- Células de la trompa de Falopio
- Células del endometrio
5. Transferencia embrionaria
En la cavidad uterina por vía transcervical
En las trompas de Falopio
6. Congelación y descongelación de embriones en su caso
 Día 1: El cigoto

Fertilized human egg

One-cell embryo, also

called a zygote

A fertilized egg at high magnification

Male and female genetic material (DNA) is in the 2
pronuclei (circles) in the center. A polar body is seen at
one o'clock - just under the shell

The (ovarian) cumulus cells around the egg have been

stripped off
This is what we see at the "fertilization check" the
morning after an egg retrieval
 Día 2: Embriones humanos de 4 células

High quality 4 cell embryo from IVF

Upper cell in middle is out of plane of focus
Zona pellucida (shell) visible as halo (not in focus)
Numerous sperm are visible attached to the outside
of the zona (they lost the race)
Embryo transfer resulted in pregnancy
Another very good 4-cell embryo (different patient)
Cumulus cells from the ovary are seen at 6 to 8 o'clock
Embryo transfer resulted in pregnancy
 Día 3: Embriones humanos de 8 células
High quality 8-cell embryo from in vitro fertilization
4 cells are seen in the plane of focus
We are hatching this embryo just prior to the embryo
transfer procedure
The holding pipette is on the far left
Another high quality 8-cell embryo
6 cells are seen in the plane of focus
Another cell is in the center, above the plane of focus -
with a little imagination you can see it
 Día 4: Embrión humano en estadío de mórula

High quality day 4 embryo from IVF

This is a compacting morula
There are many cells and the cell
borders are becoming fuzzy as the
embryo "compacts"

A morula contains about 10 - 30 cells or so. The morula stage is the final stage prior to formation of a fluid filled cavity called
the blastocoel cavity. Once the cavitation is recognizable we call the embryo an early blastocyst.

Embryo arrest at the morula stage is not uncommon, which is one reason that transfer at the blastocyst stage can be a beneficial
IVF treatment strategy.
Día 5: Embrión humano en estadío de blastocisto

Day 5 embryo
High quality human blastocyst
Técnicas de co-cultivo embrionario
 FIV con co-cultivo embrionario (1)

IVF embryos on endometrial cell coculture using cells

from the mother's own uterine lining
These day 3 embryos are being "cocultured" with the
endometrium cells
Endometrial cells are in the background - forming a
monolayer on the bottom of the culture flask
3 human embryos in coculture on a proliferating
monolayer of tubal cells
The bare plastic bottom of the culture dish is seen
between the cells
FIV con co-cultivo
embrionario (2)

More detail of tubal cells and one embryo

3 embryos with bovine tubal cell coculture

Embryos look fuzzy because plane of focus
is on the tubal cells

Tubal cell monolayer

as used for coculture
 FIV con co-cultivo embrionario (3)

Co-cultivo con transferencia en fase de blastocisto

Mouse embryos from coculture

Hatching blastocyst at bottom left
Completely hatched blastocyst at upper right detail of tubal cells and one embryo
 Fases de la fecundación in vitro (FIV)

1. Estimulación del ovario con hormonas

2. Extracción de ovocitos
3. Inseminación de los ovocitos obtenidos
Inseminación clásica
Inyección intracitoplasmática de espermatozoides (ICSI)
4. Cultivo in vitro hasta embrión en diferentes estadíos de desarrollo
Cultivo durante tres días en el laboratorio
Co-cultivo embrionario durante 6 días hasta el estadío de blastocisto
- Células de la trompa de Falopio
- Células del endometrio

5. Transferencia embrionaria
En la cavidad uterina por vía transcervical
En las trompas de Falopio
6. Congelación y descongelación de embriones en su caso
 Transferencia embrionaria

During the embryo transfer procedure the Wallace catheter is seen in the cervix and uterine lining (look below the yellow lines).
It is seen coming from the vagina into the cervix on the right. The catheter is also seen inside the uterine lining (to left side). The
embryos are released from the catheter tip - which is seen just to the right of the "L". Notice that the angle between the cervix
and uterus (green lines) is not severe. Cervix = C, Bladder = B, Lining = L, Vagina = V
 La línea endometrial

Ultrasound images of a properly prepared receptive uterine lining - endometrium

In this case the endometrial lining is 11.5 mm thick
Image on right shows uterus outlined in blue and "triple stripe" endometrial lining outlined in yellow
The endometrium is the landing pad and implantation zone for the embryos
FIV con cultivo hasta la fase de blastocistos y
transferencia uterina

2 blastocysts that were transferred with a pregnancy resulting

Transferring 2 blastocysts can yield high pregnancy rates and (almost) no risk for triplets
 Escape de la zona pelúcida
A blastocyst starting to hatch from its shell - lower right of photo
After hatching, the embryo can implant in the uterine lining
Photo taken a few minutes prior to embryo transfer
Hatching mouse blastocyst stained with fluorescent dye
Embryo was cultured on human tubal cells (coculture)
Red-stained cells (trophectoderm) - would become
placenta
Blue-stained cells are inner cell mass - fetal cells 6
"hatching" blastocyst
It is almost completely hatched out of its shell, or zona
pellucida (zona is at lower left of image)
After the blastocyst hatches, it begins to invade into the
uterine lining to "implant"
The dark areas at the top and lower right of this image are
shadows in the drop of culture media
Escape de la zona pelúcida (hatching) asistido

An 8-cell embryo in the process of assisted hatching

Holding pipette on left, hatching needle on right

As the shell (or zona pellucida) around the embryo is
dissolved, the needle is advanced to the left until a small
opening is made
 Transferencia en fase de blastocisto

One blastocyst embryo

transferred on day 5
 Transferencia en fase de blastocisto

One blastocyst embryo Nick arrived 9 months later

transferred on day 5
Pregnancy rates with blastocyst culture and transfer
 Fases de la fecundación in vitro (FIV)

1. Estimulación del ovario con hormonas

2. Extracción de ovocitos
3. Inseminación de los ovocitos obtenidos
Inseminación clásica
Inyección intracitoplasmática de espermatozoides (ICSI)
4. Cultivo in vitro hasta embrión en diferentes estadíos de desarrollo
Cultivo durante tres días en el laboratorio
Co-cultivo embrionario durante 6 días hasta el estadío de blastocisto
- Células de la trompa de Falopio
- Células del endometrio
5. Transferencia embrionaria
En la cavidad uterina por vía transcervical
En las trompas de Falopio

6. Congelación y descongelación de embriones en su caso

Criopreservación de embriones

4 blastocysts that were "left over" after the embryo transfer

These 5 day old embryos are candidates for cryopreservation
(freezing) for future use
Programa de donación de oocitos
 Receptoras de oocitos (1)
A. Grupo 1

a. Fallo ovárico primario

- Disgenesia gonadal (S. Turner, S. Swyer, disgenesia gonadal pura)
- Síndrome de Savage o del ovario resistente

b. Fallo ovárico prematuro

- Factores hereditarios: portadoras del síndrome de X-frágil, etc.
- Alteraciones enzimáticas: galactosemia, deficiencia de 17 α-hidroxilasa, defecto en la
secreción de gonadotropinas
- Trastornos autoinmunes: MEN o síndrome de neoplasias endocrinas, síndrome de
Addison, diabetes mellitus, hipotiroidismo, anticuerpos antiovario, etc.
- Factores infecciosos: parotiditis, rubéola
- Ambientales: tabaquismo, etc.
- Castración quirúrgica: por quistes o tumores ováricos
- Quimioterapia o radioterapia previa

c. Menopausia
 Receptoras de oocitos (2)
B. Grupo 2
a. Anormalidades genéticas; enfermedades transmisibles a la descendencia. Se debe realizar un
consejo preconcepcional para dilucidar las posibilidades de transmisión a la descendencia:
- Autosómicas dominantes: alopecia familiar, epidermiolisis bullosa, etc.
- Autosómicas recesivas: que comparte el varón y no acepta el uso de semen de donante:
fibrosis quística, etc.
- Enfermedades ligadas al sexo: hemofilia, etc.

b. Anormalidades cromosómicas
- Mosaicismos
- Translocaciones
- Mujeres portadoras del síndrome del X- frágil
- Inversiones cromosómicas
- Deleciones, etc.

c. Mujeres con fallos repetidos en fecundación in vitro:

- Bajas respondedoras: no responden a la estimulación ovárica
- Fallo de fecundación en repetidas ocasiones con ICSI
- Fallo repetido de implantación de los embriones (fallo de gestación repetido)
- Mala calidad oocitaria
 Receptoras de oocitos (3)

B. Grupo 2

d. Abortos de repetición
- Mala calidad ovocitaria
- Alteración cromosómica en la mujer
- Alteración cromosómica en los embriones

e. Ovarios inaccesibles para la obtención de ovocitos

- Pelvis congeladas
- Existencia de múltiples adherencias
- Etc.

f. Mujeres mayores de 40 años con ciclo ovárico normal

 Infertilidad: Volumen del ovario y número de
folículos ováricos
Low ovarian volume and low antral follicle counts

The right ovary from the same woman

The left ovary is outlined in blue and is small (low volume) This ovary is also small with only 2 antral follicles
Only 1 antral follicle is present She had a total of 3 antrals from both ovaries
This woman had regular periods and a normal day 3 FSH test Attempts to stimulate her ovaries for IVF were not successful
 Donación de oocitos
Requisitos de las donantes

• Edad: 18 a 35 años
• Buen nivel intelectual
• Historial familiar negativo para enfermedades de transmisión genética
• Cariotipo normal (estudio cromosómico)
• Estudio negativo para enfermedades de transmisión sexual
SIDA
Hepatitis B y C
Clamydia
Herpes virus
Citomegalovirus
Toxoplasma gondii
Rubeola
Sífilis
• Normalidad del aparato reproductor
• Salud física y mental
• Historia de fertilidad previa y/o adecuada respuesta al tratamiento de estimulación ovárica
Recién nacidos vivos en función de la edad de la mujer
receptora de los embriones

It shows the rate of live births per embryo transfer procedure by the age of the recipient of the embryos. The blue line shows data using the infertile
woman's own eggs for IVF, while the black line shows IVF data from using donor eggs.
This chart is very useful in illustrating the decline in live birth rates by female age beginning at about age 31. This curve becomes steeper (egg quantity and
quality decreasing at a faster rate) starting at about age 37. It is very important to remember that each data point on the curve below represents an average
live birth rate from many cases. Every couple is unique and could be more fertile, or less fertile as compared to the average for their age.
Another interesting point that is illustrated here is that there is no decline in live birth rates by age of recipient when donor egg IVF is being utilized. In
other words, the age of the eggs is very important, but the age of the uterus is not.
 El factor masculino:
Laboratorio de Andrología
 Laboratorio de Andrología

- Análisis básico del semen

- Congelación del semen

- Estudio de los marcadores de supervivencia espermática

tras la congelación a bajas temperaturas

- Marcadores moleculares de fertilidad

- Fragmentación del ADN

- Análisis por citometría de flujo del semen

- Lavado de semen y detección de VIH y Hepatitis C en

muestras de pacientes infectados
 Análisis del semen
Conocimiento del potencial fértil del varón

o Concentración de espermatozoides

o Movilidad
La concentración y movilidad de los espermatozoides en las muestras son
valoradas con la ayuda de un microscopio de contraste de fases a 20
aumentos, en una cámara especial en la que no se afecta el comportamiento
de los espermatozoides

o Morfología
Tras realizar una tinción histológica adecuada, la muestra es analizada a
100 aumentos, en busca de defectos de los espermatozoides en la cabeza,
la pieza intermedia y la cola

o Viabilidad
Porcentaje de espermatozoides viables en el eyaculado
Estudio de los espermatozoides

Fixed and stained human sperm images from a IVF lab

Some abnormal sperm morphology is shown
Very high magnification
Sperm morphology scoring tells us what proportion of the sperm are
shaped normally
Very low morphology scores can indicate male infertility
Because not all technicians use exactly the same criteria for "normal"
sperm, morphology percentages will vary significantly between
laboratories (it is a subjective assessment)
Estudio de los espermatozoides

Sperm in a counting chamber (hemocytometer)

This is the system to determine sperm counts (concentration) and motility percentages
 Pruebas adicionales en el semen

o Ensayos inmunológicos
Detección de la presencia de anticuerpos antiespermatozoide en semen y
suero de la pareja, de los que se ha demostrado una relación directa con la
infertilidad

o Ensayos bioquímicos
Marcadores de glándulas accesorias en el aparato genital masculino, cuyo
funcionamiento es fundamental en la correcta maduración de los
espermatozoides

o Capacitación diagnóstica
Mediante esta técnica se consigue obtener el mayor número de
espermatozoides móviles posible, así como el lavado del plasma seminal
(perjudicial para los espermatozoides)
Eliminación del virus de la hepatitis C y del VIH

Eliminación del virus de la Hepatitis C en hombres Lavado de semen en varones seropositivos para el
portadores mediante lavado de semen virus de inmunodeficiencia humana
 Recuperación Espermática Extrema

Características
Técnica empleada en el tratamiento de pacientes azoospémicos, es decir,
pacientes que carecen de espermatozoides en el eyaculado

Obtención de espermatozoides directamente del testículo o del epidídimo

Técnica biópsica
Extracción de una pequeña muestra de tejido testicular

Requisitos
Existencia de espermatozoides en número mínimo

Indicaciones principales
Azoospermia obstructiva
Azoospermia no obstructiva o secretora
Imposibilidad de recoger la muestra
Inmovilidad absoluta de espermatozoides en el eyaculado
 Banco de semen
Las muestras de semen se conservan congeladas, usando como refrigerante
nitrógeno líquido, técnica que se conoce como criopreservación

Pacientes

- Congelaciones pre-vasectomía
- Pacientes sometidas a tratamientos de reproducción asistida cuyas
parejas, por motivos laborales, no puedan estar presentes en el
momento en el que se necesita su semen
- Congelaciones previas a tratamientos de quimio- o radioterapia
- Dificultades en la obtención del eyaculado
- Muestras de muy mala calidad
- Biopsias testiculares
- Aspiraciones de epidídimo
 Banco de semen

Donantes

Todos los donantes, tras exploración física y anamnesis completa

incluyendo historial familiar, son testados para:

- Antígenos de la Hepatitis B
- Anticuerpos anti-HIV
- Chlamydia
- Anticuerpos anti-herpes virus
- Sífilis
- Anticuerpos anti-Hepatitis C
- Gonorrea
- Citomegalovirus
 Utilización de semen de donante

La utilización de semen de donante en las técnicas de reproducción

asistida está indicada como tratamiento de la infertilidad en los
siguientes casos:

Æ Parejas heterosexuales con ausencia de espermatozoides

tanto en el eyaculado como directamente en el testículo
y/o epidídimo

Æ Ante la posibilidad de transmisión de trastornos genéticos

o enfermedades contagiosas al utilizar semen conyugal

Æ Enfermedades inmunitarias documentadas

Æ Mujeres sin pareja

Diagnóstico genético preimplantacional
 Diagnóstico genético preimplantacional

Análisis cromosómico o genético

Las técnicas utilizadas para este propósito son dos, en

función de la indicación:

Æ Fluorescencia con hibridación in situ (FISH)

si se trata de anomalías cromosómicas

Æ Reacción en cadena de la polimerasa (PCR)

en el caso de anomalías de origen génico
Biopsia embrionaria
Consiste en extraer 1 ó 2 células de un embrión, mediante técnicas de micromanipulación. Para ello, se realiza un pequeño
orificio en la cubierta externa del embrión, la zona pelúcida, lo cual permite introducir una fina micropipeta (35 micras) para
aspirar la célula embrionaria. La célula extraída es procesada para su posterior análisis genético o cromosómico y el embrión
prosigue su desarrollo embrionario normal.
Biopsia embrionaria
 Fluorescencia con hibridación in situ (FISH)

Técnica
Marcaje con fluorescencia de los cromosomas con sondas de ADN específicas
para los cromosomas motivo de estudio

Identificación del número de copias existentes de un cromosoma determinado

Aplicaciones

Análisis de anomalías cromosómicas numéricas (aneuploidías) como, por

ejemplo, la presencia de 3 cromosomas 21, responsable del síndrome de
Down y otras anomalías numéricas que originan abortos de repetición o
descendencia con cromosomopatías

Estudio de anomalías cromosómicas estructurales, sobre todo translocaciones.

En padres portadores de una alteración de este tipo, se puede seleccionar para
la transferencia embriones normales o equilibrados

Identificar los cromosomas sexuales X e Y y así determinar el sexo de los

embriones en enfermedades ligadas al sexo (hemofilia, distrofia muscular
de Duchenne, etc.)
 Fluorescencia con hibridación in situ (FISH)

Hibridación in situ fluorescente (FISH). Consiste en marcar con fluorescencia los cromosomas con sondas de ADN (ácido
desoxiribonucleico) específicas para los cromosomas motivo de estudio. A continuación, con el microscopio de fluorescencia,
podemos identificar el número de copias para un cromosoma determinado.
FISH

Análisis de amniocitos mediante FISH

con sondas para los cromosomas 18 (CEP
18 Spectrum Aqua), X (CEP X Spectrum
green) e Y (CEP Y Spectrum Orange)
(Vysis).
Se observa un cromosoma X (señal
verde), un cromosoma Y (señal roja) y
tres copias para el cromosoma 18. Esta
célula corresponde a un feto masculino
con trisomía 18.
 Reacción en cadena de la polimerasa (PCR)

Técnica
Consiste en la amplificación de secuencias específicas del ADN de un gen,
en las que la presencia de una mutación desencadena una enfermedad de
origen génico

Permite diferenciar qué embriones son normales y cuáles tienen un gen

mutado y, por tanto, desarrollarán la enfermedad

Aplicaciones
Se han descrito aproximadamente 5.000 enfermedades de origen génico,
como son:
Fibrosis quística
Distrofia miotónica
Enfermedad de Tay-Sachs
Síndrome de Marfan
Beta-talasemia
Anemia falciforme
Enfermedad de Huntington
Etc.
Reacción en cadena de la polimerasa (PCR). Consiste en la amplificación de secuencias específicas del ADN de un gen, en las que la
presencia de una mutación desencadena una enfermedad de origen génico. Nos permite diferenciar qué embriones son normales y cuáles
tienen un gen mutado y, por tanto, desarrollarán la enfermedad.
 Enfermedades humanas detectables mediante
diagnóstico pre-implantacional
Autosómicas recesivas
Fibrosis Quística (gen CFTR)
Atrofia Muscular Espinal (Tipo I o de Werdnig-Hoffmann) (gen SMN1, Deleción exon 7)
Talasemia (25-26delAA, IVS2+1G>A, IVS1+6T>C, IVS1+110G>A)
Anemia Falciforme
Incompatibilidad factor Rhesus D. (Duplex RhD y RhCE)

Autosómicas dominantes
Distrofia Miotónica o Enfermedad de Steinert
Enfermedad de Huntington
Enfermedad Charcot-Marie-Tooth (gen MPZ)
Enfermedad AD Riñón poliquístico
Síndrome de Marfan (MFS) (gen fibrillin-1 [FBN1])

Ligadas al cromosoma X
Síndrome del X frágil
Distrofia Miotónica de Duchenne (DMD) y DM Becker (DMB)
Hemofilia A
Detección de anomalías cromosómicas

Dotación cromosómica 47,XY,+21

Diagnóstico prenatal de Síndrome de Down en líquido amniótico
Detección de anomalías cromosómicas

Dotación cromosómica 47,XXY

Alteraciones de los óvulos y embriones
 Alteraciones de los óvulos

This very immature egg is at the "germinal vesicle" stage

A "good" egg from a 32 year old woman It will not mature and fertilize in the IVF lab
The germinal vesicle is seen as the circular structure inside
the egg at 8 o'clock
 Oocitos con las células del cumulus en el
momento de su obtención para FIV

Egg (dark area in center) in the middle of it's adherent mass

of hundreds of cumulus cells Closer view of an egg (bottom left area) in the cumulus
Cumulus cells are from the ovarian follicle Egg appears dark here because little light penetrates the
They stick to the egg when it releases from the follicle cumulus mass
 Oocitos inmaduros

Egg, also called oocyte is in center of picture

Many cumulus cells from the ovary are seen around the egg Another low quality, oocyte from a woman over 40
This is a low quality, oocyte from a woman 41 years old Polar body is visible at 10 o'clock
Egg is irregularly shaped and dark
 Oocitos anormales

An egg with a severely fragmented polar body An egg with many pronuclei (hard to see here)
at 10 to 1 o'clock and severe fragmentation
This is quite unusual This is rare and consistent with a chromosomal
abnormality
 Oocitos anormales

An egg with numerous vacuoles (look like moon craters)

Another rare finding - cause unknown This egg has no zona pellucida (shell) around it
This woman is in her 20's and all of her eggs had this Egg is on the lower left, cumulus cells from the ovary are
appearance stuck to it on the right
This woman's eggs had other unusual abnormalities as well
 Oocitos degenerados

3 degenerative eggs Close detail of one of the degenerative eggs from the same
This patient also had some "normal" eggs case as shown above
Óvulo humano fertilizado anormal
Presencia de tres pronúcleos

3 pronuclei are seen in the center of the cell

Each pronucleus contains 23 chromosomes
This embryo has 69 chromosomes instead of the normal 46
Zona pellucida (shell) is visible as a halo around the periphery
Sperm are visible at one and 8 o'clock on the outside of the zona
 Embriones multinucleados

Fertilized one cell embryo

Multiple pronuclei are visible (normal is 2)
This abnormal 2-cell embryo is from IVF from a 41
Embryo is chromosomally abnormal (has too many
year-old woman
chromosomes)
Multiple nuclei (circular structures that look like
Numerous sperm are visible bound to the zona, outer
moon craters) are seen in both cells
shell of the embryo
Chromosomally abnormal embryos such as this are
Chromosomally abnormal embryos such as this are not
not transferred to the mother's uterus
transferred to the mother's uterus
 Embriones multinucleados

Normal Abnormal
Chromosomes line up in Chromosomes line up
straight line on spindle erratically

D.E. Battaglia, et al. (1996) Influence of maternal age on meiotic spindle assembly in
oocytes from naturally cycling women. Human Reproduction, Vol. 11:2217-2222.
 Oocitos y embriones humanos con alteraciones en
la zona pelúcida

"Empty zona" - the cells of the This is a 6-cell embryo of high

This egg has no zona pellucida around it
Egg is on the lower left, cumulus cells
from the ovary are stuck to it on the right
This woman's eggs had other unusual
abnormalities as well
embryo have escaped through
a crack in the zona (shell)
The break can be seen by the
"X" at 5 o'clock
This zona pellucida is slightly
thicker than average
quality (grade)
The zona pellucida is
irregularly shaped and thicker
than normal
The irregular shape is best
seen in the 3 to 6 o'clock area
 Embriones humanos con alteraciones en la
zona pelúcida

This is a 4-cell embryo of high quality

A high grade 4-cell embryo The zona pellucida is thicker than normal The zona here is unusually thin
with a normal zona Assited hatching was done prior to
embryo transfer
 Blastocistos de baja calidad

Blastocyst showing poor morphology in cells of both

components - the inner cell mass (at 6 to 9 o'clock) and the Poor quality day 6 blastocyst
trophectoderm (the rest of the visible cells) Many of the cells are necrotic (dead and disintegrating)

These low quality blastocysts are shown to indicate that blastocyst culture is not a magic bullet for infertility. Most human embryos
do not have the genetic potential to hatch from their shells, implant and continue normal development in order to result in a live birth.
4. Clonación
Clonación
Transferencia nuclear
somática

Freddy
Figure 4.5. Procedure for transplanting blastula nuclei into activated
enucleated Rana pipiens eggs. The relative dimensions of the meiotic
spindle have been exaggerated to show the technique. "Freddy," the
handsome and mature R. pipiens in the photograph, was derived in this
way by M. DiBerardino and N. Hoffner Orr. (Gilbert, 2003).
 Transferencia nuclear somática

Figure 4.6. Percentage of successful nuclear transplants as a function of the developmental age of the donor nucleus.
The abscissa represents the developmental stage at which a donor nucleus (from R. pipiens) was isolated and inserted
into an activated enucleated oocyte. The ordinate shows the percentage of those transplants capable of producing
blastulae that could then direct development to the swimming tadpole stage. (Gilbert, 2003).
 Clonación
Clonación a partir de núcleos de
células diferenciadas

Donante del núcleo: Renacuajo hembra

en estadío de esbozo caudal con padres
albinos

Donante del oocito: Hembra tipo salvaje

Resultado: ranas hembras albinas

Figure 4.7. A clone of Xenopus laevis frogs. The nuclei for all
the members of this clone came from a single individual a
female tailbud-stage tadpole whose parents (upper panel) were
both marked by albino genes. The nuclei (containing these
defective pigmentation genes) were transferred into activated
enucleated eggs from a wild-type female (upper panel). The
resulting frogs were all female and albino (lower panel).
(Gilbert, 2003).
 Clonación de
mamíferos

Figure 4.8. Cloned mammals, whose nuclei

came from adult somatic cells. (A) Dolly, the
adult sheep on the left, was derived by fusing a
mammary gland cell nucleus with an
enucleated oocyte, which was then implanted
in a surrogate mother (of a different breed of
sheep) who gave birth to Dolly. Dolly has
since produced a lamb (Bonnie, at right) by
normal reproduction. (B) Cloned mice and
their "parents." The upper left black mouse is
the oocyte donor, while the upper right brown
(agouti) mouse is the nucleus donor. The white
mouse in the lower row is the mouse into
whose uterus the resulting embryos were
implanted. The two agouti mice beside her are
cloned mice, derived from the injection of the
agouti nucleus into the oocyte of the black-
furred parent (C) Procedure used for cloning
mice. (A photograph by Roddy Field, © Roslin
Institute; B courtesy of T. Wakayama and R.
Yanagimachi.) (Gilbert, 2003).
Clonación de Dolly

Figure 21.11. A sheep called Dolly was the

outcome of the first successful attempt at animal
cloning. (A) Experimental strategy used by Wilmut
et al. (1997). During culture in serum-depleted
medium the normal cell cycle is interrupted and
cells move from the G1 phase of cell growth to a
quiescent stage, G0 where there is no cell division.
This quiescent state was thought to facilitate
successful nuclear transfer (see text). Note that in
the Wilmut et al. (1997) study the nuclear transfer
occurred by cell fusion, but in other cases different
strategies have been used. For example, in the
successful cloning of adult mice reported by
Wakayama et al. (1998), a very fine needle was
used to take up the donor cell nucleus with minimal
contamination by donor cell cytoplasm. The donor
cell was quickly, but very gently, microinjected into
the enucleated oocyte. (B) Dolly with her first born,
Bonnie. Original photo kindly provided by the
Roslin Institute.
Procedimiento general
de obtención de
ratones transgénicos
 Inserción de DNA en una célula embrionaria

Figure 4.18. Insertion of new DNA into embryonic cells. Here, DNA (from cloned genes) is injected into the a pronucleus
of a mouse egg. (Gilbert, 2003).
 Obtención de animales transgénicos

Figure 4.10. Production of transgenic sheep. The structural gene for an important human protein (such as clotting factors, insulin, or
a1-antitrypsin) is linked to the regulatory region (promoter) of a sheep milk protein gene (such as that for casein or lactalbumin). This
recombinant gene is injected into the pronucleus of a newly fertilized sheep egg, and the egg is implanted into a foster mother. The
newborn sheep are screened for the presence of the human gene. When the transgenic sheep mature, the human gene should be
expressed in the mammary gland and the protein secreted into the milk. From the milk one can then isolate large amounts of the
protein for pharmaceutical use. (Gilbert, 2003).
 Prometea
El primer caballo clonado del mundo
C. Galli, 28-5-2003

El embrión procede de células

extraídas a su propia madre
Pieratz-Cryozootech-Stallion
El segundo caballo clonado
del mundo
Cryozootech/LTR-CIZ, 25-2-2005

Clon de Pieratz, campeón

mundial de resistencia
 Clonación de terneros
Objetivo: producción de anticuerpos humanos
Nature Biotechnology, 2002
 Pampa
El primer clon de una vaca en Argentina
BioSidus, 2002

Objetivo: creación de proteínas humanas con fines terapéuticos

 Copycat
Primer gatito nacido
mediante clonación

College Station University

(Texas), 2002
El regreso del Tigre de Tasmania
Reproducción de genes
Mike Archer. Museo de Australia, Sidney, 2002
 ANDi
Primer mono rhesus genéticamente modificado (gen GFP)

Science, 2000
Oregon Primate
Research Center

Objetivo: crear nuevos modelos animales para el estudio y tratamiento de enfermedades humanas
 Noel, Angel, Star, Joy, Mary
Primeros cerditos creados para realización de xenotransplantes
Alan Colman, PPL Therapeutics, Virginia, 25-12-2001
Células madre y clonado terapéutico
Preparación de células madre embrionarias
Clonación reprodutiva y terapéutica

Figure 8-6. Reproductive and therapeutic cloning. (Alberts et al., 2008).

Terapia
con
células
madre

Figure 4.22. ES cell therapeutics. (A) Human

embryonic stem (ES) cells can be derived from
the inner cell mass of blastomeres or from
human germ cells before they initiate meiosis.
These cells can differentiate in culture to form
the more restricted stem cells for neural, blood,
heart muscle, or lymphocyte lineages. (B) The
differentiation of ES cells into lineage-restricted
(neuronal and glial) stem cells can be
accomplished by altering the media in which the
ES cells grow. (Gilbert, 2000).