Q.1. Describe oogenesis in detail.

Ans :

 The process of maturation and differentiation of PGC to oogonia, primary oocytes,

secondary oocytes and mature ova in the female genital tract is oogenesis.
 Location: Ovarian cortex.
 Features of oogenesis :
1. Starts before birth (10th week)
2. Stops in the middle (birth to puberty)
3. Restarts at puberty (11–13 years)
4. Continues up to menopause (45–55 years)
 Processes of oogenesis :
1. Mitosis
2. Meiosis
3. Growth of follicles
4. Differentiation of follicles
 The ovarian cortex contains many large round cells called “oogonia”.
 The oogonia pass through the stages of primary and secondary oocyte and ovum.

Stages of oogenesis
 Q.2. Explain oocyte activation.

Ans :

Activation of oocyte includes following stages :

 The sperm has to pass through three barriers (Corona radiata, Zona pellucida
and Vitelline membrane) which ultimately activates the oocyte.
 The acrosome enzyme of sperm ; hyaluronidase digests the cells of corona
radiata and sperm penetrates the Corona radiata.
 The glycoproteins on the outer surface of sperm head binds with
glycoproteins of Zona pellucida by binding to Zp3 and Zp2 receptors.
 Acrosin released by sperm causes digestion of ZP around sperm head.
 The plasma membrane of oocyte releases lysosomal enzyme which alters
zona pellucida and this reaction is called “zona reaction”.
 Zona reaction prevents polyspermy.
 Spermatozoa passes the third barrier; Vitelline membrane where there is a
fusion of plasma membrane of two cells at receptor sites.
 This fusion is initiated by disintegrin peptides from sperm head.
 The contact of sperm with vitelline membrane triggers the calcium wave
activation (depolarization) of oocyte cytoplasm. This triggers :
1. Second meiotic division of secondary oocyte
2. Contact of cortical granules with plasma membrane and release of
lysosomal enzymes from cortical granules for prevention of
polyspermy.
3. Alterations in plasma membrane of oocyte and zona pellucida for
prevention of entry of other spermatozoa in oocyte.
4. Ultimately activation of oocyte.
Q.3. Write a note on fusion of sperm and oocyte.
Ans :
 Prior to the fusion of sperm and oocyte an important event takes place ;
“capacitation of sperm”.
 Capacitation is the maturation of spermatozoa.
 It includes changes in the sperm cell membrane and signal transduction
(calcium influx, release of acrosomal enzymes, etc.). It also includes major
changes in surface glycoproteins; Removal of glycoproteins and exposing
zona binding proteins.
 The process includes following stages :
 Acrosome reaction in which capacitated sperm head establishes multiple
contacts between plasma membrane and outer membrane of acrosomal cap
and discharges chemical substances that facilitate penetration of barriers
around oocyte.
 Acrosome reaction leads to release of acrosomal enzymes hyaluronidase,
protease, acrosin and acid phosphatase which facilitates the penetration of
zona pellucida.
 Disintegration of barriers including Corona radiata, Zona pellucida and
Vitelline membrane through sperm has to pass for successive activation of
oocyte and completion of fusion.
 Sperm penetrates corona radiata with the help of enzyme hyaluronidase.
 The glycoproteins on the outer surface of sperm head binds with
glycoproteins on the zona pellucida of ovum. Due to release of lysosomal
enzymes by plasma membrane of the oocyte zona pellucida alters and results
in “zona reaction” which prevents polyspermy and spermatozoa penetrates
zona pellucida.
 Spermatozoa passess the third barrier ; Vitelline membrane where there is a
fusion of plasma membrane of two cells at receptor sites.
 Calcium wave in oocyte is triggered in oocyte cytoplasm by above fusion.
 It triggers second meiotic division of secondary oocyte, prevention of
polyspermy due to release of lysosomal enzymes from cortical granules,
alteration of oocyte plasma membrane, zona pellucida which prevents entry
of other spermatozoa in oocyte and egg maturation.
 Nuclear fusion involves entry of both head and tail of spermatozoa in oocyte
cytoplasm without plasma membrane.
 It leads to the completion of second meiotic division and release of ovum
with 1N DNA and second polar body. Reconstitution of oocyte chromosomes
results into female pronucleus and sperm head makes a rotation of 180°
within the oocyte cytoplasm , transforms into a male pronucleus.

Fusion of sperm and oocyte

Q.4. Explain post fertilization changes in oocyte.
Ans :
 sperm enters in the cytoplasm of oocyte with its head and tail without
plasma membrane and fusion of sperm and oocyte takes place.
 After the fertilization of gametes, approximation of pronuclei takes place
near the middle of cytoplasm of ovum.
 After the entry of sperm in cytoplasm of oocyte, oocyte immediately
completes its second meiotic division and releases the ovum with 1N DNA
and second polar body.
 Reconstitution of oocyte chromosomes takes place and it forms female
pronucleus.
 Within the cytoplasm of oocyte sperm head makes 180° rotation and
transform into male pronucleus.
 Male and female pronuclei are made of one chromatid.
 Replication of DNA takes place to form a second chromatid in each
chromosome (1N–2N DNA).
 Two centrioles appear and disappearance of nuclear membranes resulting in
splitting of each chromosome into two occurs and ovum transforms into
“zygote”.
Q.5. Write a note on syngamy.

Ans :

 When the male and female pronuclei are formed, after few hours they
migrate to the center of the oocyte.
 During this period DNA is synthesized for the preparation of first mitotic
division.
 When the mitotic metaphase spindle formation takes place, it involves
duplication of the proximal centriole contributed by the sperm centrosome.
 It leads to the formation of a pair of polar centrioles, and the chromosomes
are aligned along the spindle equator.
 The plane of cell division is mediated by astral microtubules that extend from
the mitotic spindle to the plasma membrane.
 In 18 to 24 hours, after the gamete fusion two sets of chromosomes come
together called as syngamy in which a cleavage furrow is formed when first
mitotic anaphase and telophase are completed and the zygote becomes a
two cell embryo.
Q.6. What is IVM? Explain in detail.

Ans :

 IVM is the maturation of immature oocytes in culture after their recovery

from small antral follicles at the stage prior to selection and dominance.
 It requires very little or no FSH (stimulation).
 It reduces primary adverse effects caused by ovarian hyperstimulation ;
ovarian hyperstimulation syndrome.
 It is a good option for cases for which no standard protocol is suitable like
FSH resistance, contraindications for ovarian stimulatory drugs, and the need
for urgent fertility preservation.
 Indications for IVM :
1. Women at risk of OHSS
2. PCOS or PCO-like ovaries
3. Those with estrogen-sensitive cancers
 IVM for fertility preservation :
1. Oocyte cryopreservation is the promising method for female fertility
preservation.
2. IVM can be performed urgently irrespective of the phase of the
menstrual cycle without affecting the quantity and maturation rate of
the oocytes.
 Q.7. Write a note on oocyte handling.

Ans :

 After the aspiration of follicular fluid oocytes are screened from the follicles.
 identification of the cumulus–oocyte complexes and evaluation of their
maturity are carried out.
 Prior to ICSI, cumulus cells are removed by denudation of the mature oocytes
[Enzymatic (enzyme hylase) and mechanical treatment] as it may block the
injecting needle, during oocyte microinjection.
 Oocyte maturation is determined morphologically by the absence of the GV
and the presence of the first polar body.
 For the preparation of mature oocytes for ICSI appropriate conditions are
maintained :
1. constant pH of 7.3 and a stable temperature of 37°C.
2. 4-(2-hydroxyet4hyl)-1-piperazineethane sulfonic acid (HEPES)-
buffered culture media are used.
3. The correct temperature is maintained by the use of a microscope
equipped with a heated stage.
4. procedures are performed with Earle’s balanced salts solution and
CO2-equilibrated paraffin/mineral oil that prevents evaporation of the
medium and minimizes the fluctuations of both the pH and the
temperature.
5. The temperature fluctuations should be avoided as it may leads to the
changes including reduction in spindle size, disorganization of
microtubules within the spindle, and even a complete absence of
microtubules.