Gametogenesis & fertilization

Lectured by

รศ.สิทธิชัย เอี่ยมสะอาด (Associate. Prof. Sitthichai Iamsaard, Ph.D.)

Department of Anatomy, Faculty of Medicine, Khon Kaen University

Official email: sittia@kku.ac.th 1

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Objectives

1. To know features of spermatogenic and egg cells

2. To describe and compare the events of

spermatogenesis and oogenesis

3. To understand the ovarian follicle development and

ZP structure

4. To explain the fertilization process, sperm

capacitation, acrosome reaction, and egg activation

5. To distinguish the fertilized egg from unfertilized one

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Resource: http://desertbruchid.net/4_GB1_LearnRes_fa10_f/4_GB1_LearnRes_Web_Ch10.html

1.Sexual interaction and intercourse

2. Internal fertilization

3. Gestation period (~ 9 months) 4. Childbirth

Involves:

- Creation of genetically novel individual

Requires: Gametogenesis - Spermatogenesis & Oogenesis

Sexual reproduction of human

-Sexual intercourse b/w a man and a woman

- Unity of sperm & oocyte at fertilization

The Human Life Cycle

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Note: Missionary Position

General structure and function of human reproductive organs

Origin of germ cells

Undifferentiate embryo at 4th weeks

Consideration of spermatogenesis (sperm forming)

At puberty (13-16 years old)

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Embryonic gonad (testis)

At week 4 of male embryo

Primordial

germ cells

(PGC)

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PGCs migrate to form gonad

Before puberty

PGCs remain dominant in testis

• PGCs differentiate into “spermatogonia” via mitosis

• Spermatogenesis plays (via meiosis) throughout male reproductive life

- produce sperm by spermatogenesis

2. Interstitial CNT

- contains Leydig cells which produce

testosterone

1.Seminiferous tubules

Microstructure of

adult testicular tissue

- Complex stratified epithelium

containing two basic cell populations:

(1) SPERMATOGENIC CELLS

(2) SERTOLI CELLS

- Non-replicating physical support cells

• LEYDIG CELLS

- Produce and release testosterone

In seminiferous epithelium

In interstitial CNT

1. Spermatogonia

Sertoli cells

4. Spermatids 5. Spermatozoa

Leydig cell

“5 spermatogenic cells”

Smooth muscle cell

2. Primary spermatocyte

3. Secondary spermatocyte

Cell members in testicular tissue

- Involved in spermatogenesis

(somatic cells)

Hormonal Control of Male Reproduction

Resource: http://philschatz.com/biology-book/contents/m44841.html

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3 PHASES

1. Spermatogonial Phase

(Mitosis)

2. Spermatocyte Phase

(Meiosis I&II )

3. Spermatid Phase

(Spermiogenesis)

Concept of SPERMATOGENESIS: formation of sperm (64 days, ~35 C)

Meiosis I (reduction division)

Meiosis II (equatorial division)

Spermiogenesis (differentiation)

DNA replication

(Diploid cell containing two sets of chromosomes (2n), one set inherited from each parent)

(embryo until puberty period)

Puberty

To Male RP life

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The “A pale & B spermatogonia” determined to be mature sperm

Resource: https://www.researchgate.net/profile/Ashok_Agarwal2/publication/259674480/figure/fig5/AS:296965855301640@1447813586084/Differentiation-of-a-human-diploid-germ-cell-into-a-fully-functional-spermatozoon.png

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Types of spermatogonia: stem cells (2N)

Primary spermatocyte

Type B spermatogonia

A dark A pale A long

12 http://journals.cambridge.org/fulltext_content/ERM/ERM3_03/S1462399401002319sup013.gif

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Pachytene primary spermatocyte: important for genetic recombination and genetic diversity

- Largest spermatogenic cells detected - Chromosomes clump during division

Leptotene: chromosome paring

Zygotene: chromosome condensation, double-strand breaks

Pachytene: synapsis and recombination begins

Primary spermatocytes (Pachytene stage)

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Spermatids differentiate to form sperm (structural maturation) via “spermiogenesis”

Secondary spermatocyte (n)

Meiosis II (equatorial division)

Meiosis I (reduction division)

A series of structurally

maturational changes of

spermatids to form sperm

14 Resource: https://www.researchgate.net/profile/Ashok_Agarwal2/publication/282917724/figure/fig7/AS:391465520910338@1470344061138/Diagrammatic-representation-of-the-steps-where-the-histones-are-replaced-with-the.jpg

Protamine used to replace for histone in packaging DNA of sperm during spermiogenesis

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How to condense sperm DNA?

Condensed nucleus (chromatins+protamines)

Summary of

Spermiogenesis

in details

1. Nucleus condenses

(chromosomes condense and nucleoplasm removed)

2. Flagellum or tail develops

3. Round spermatid elongates

4. Acrosome formed from golgi body

5. Mitochondria aggregates around base of forming flagellum

6. Mitochondria fuses to form supermitochondrion (in humans)

7. Most cytoplasm of sperm is shed and phagocytosed by Sertoli cell

1. Remove excess cytoplasm (residual body) from developing spermatid

2. Move spermatids toward the lumen of the seminiferous tubules - ectoplasmic

specializations

3. Cultivate and mediate maturation of spermatids

4. Segregate groups of developing gametes

5. Secrete fluid to transport sperm in reproductive tract

6. Secrete hormones and other factors

Summary of Sertoli cell’s function in details

Residual body

Blood-testis barrier or Sertoli cell barrier (SCB, now is a correct term)

Germinal epithelium of testis

Tight junction of

SCB

Sertoli

cell Sertoli

cell

Spermatogonia

1st Spermatocyte

2nd Spermatocyte

Spermatid

Sperm

Basal lamina

Divides the Semi. lumen into 2 compartments

Basal compartment

Adluminal compartment

-Prevent the passage of cytotoxic agents into semi. tubule.

Sperm has less cytoplasm

Human sperm structure

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“Required: the repetitive 5 mitotic divisions of spermatogonial cells

and followed by 2 meiotic divisions to gain 96 mature sperm”

How is the number of

spermatozoa increased?

Male fertility (depends on number and % motility of sperm)

- Average volume of ejaculated semen = 3.5 ml

- A concentration of about 100 million sperm/ 1 ml of semen (less than 20 million/ ml = sterile)

-Via entire “spermatogenic cycle (64 days)” of spermatogenesis

Spermatogenic cycle (~64 days) in human

1Ad

2Ad

1st Mitosis

3Ap 1Ad

2nd Mitosis

6B1

3rd Mitosis

12B2

5th Mitosis 24 primary spermatocytes

Reserved

48 secondary spermatocytes

Meiosis I

96 spermatids

Meiosis II

96 mature spermatozoa

Spermiogenesis

Reductional division (several weeks)

Equational division (about 8 hours)

4th Mitosis

Epididymis Transit

- Non-motile testicular sperm

Peristalsis

- Motile epididymal sperm

Spermatogonial mitotic divisions (5 times)

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Functional maturation &

storage of sperm produced by testes taking place at the

“Epididymis” before

ejaculation

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Fertility & capacitation factors produced in the epididymis

Associated reproductive glands of male

Epididymis

Vas deferens

- Head - Body - Tail

Ampulla

Seminal vesicle

Prostate gland

Cowper’s gland

Semen after ejaculation (ave, 3.5 ml )

Human sperm within seminal fluid/plasma observed under simple microscope

The sperm count varies from 20 to 150 million sperm per milliliter of semen.

25 Resource: http://www.tankonyvtar.hu/hu/tartalom/tamop425/0010_1A_Book_angol_05_termeleselettan/images/12a13.png

zygote

“Fertilization” takes place

• Sperm about 300-500 x 106 cells/ ejaculation (~10% abnormal sperm) will be deposited within posterior fornix of vagina

• Only 300-500 capacitated sperm can meet a mature egg at the

ampulla of oviduct and only one sperm can fertilize it.

Oogenesis occurs within ovary

Consideration of oogenesis (oocyte forming) in ovary

At puberty (12-14 years old)

Embryonic gonad (ovary)

At week 4 of male embryo

Primordial

germ cells

(PGC)

PGCs migrate to form gonad

At birth

5th month of fetal life

PGCs differentiate into oogonia Mitosis

Meiosis I Oogonia divides to be

“primary oocyte” within “primordial follicle” of ovary

- No oogonia

- Primary follicles containing

only primary oocyte - Inactive ovary

Meiosis I (prophase arrested)

“Oogenesis begins at 5th mount of fetal life”

Menopause (45-50 years old)

- No oogenesis - No menstruation

- Follicular development

- Ovulation

- Fertilization and pregnancy (if any)

- Menstruation cycle

- Primary oocyte (Meiosis I completed)

during ovulation

- Secondary oocyte (Meiosis II completed )

after fertilization

Events: Follicle development, ovulation, and corpus luteal formation

Internal Structure of Adult Ovary

Anatomy of an ovarian follicle

1. A growing (primary) oocyte (ovum)

2. Follicular (granulosa) cell 3. Basal lamina

Graafian follicle

Follicular development within ovarian cortex

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ZP is firstly formed at the “unilaminar primary follicle”

Granulosa cells produce estrogen secreting into antrum

3. Secondary (antral) follicle 4. Mature (Graafian) follicle

A huge antrum More than one small antrum

Antral fusions

Days

FSH Ovarian follicles Estrogen Stimulates Grow

Produce

Before ovulation

Ovulation

Structures of mature Graafian follicle

ZP

Secondary oocyte present “during ovulation”

Presence of the first polar body

Oocyte structure

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Secondary oocyte (arrested in metaphase II) after ovulation present the 1st polar body

Primary oocyte in ovarian follicle

38 Recourse: https://canvas.instructure.com/courses/1021937/files/44221811/preview?verifier=xucOaEcu6uBIK5qikJZq8hUFzMSY4MoB6JaUxqce

Oogenesis

Vs

Follicular development

Perivitelline space

Secondary oocyte (completed

metaphase II ) or fertilized egg

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Summary of stages in oogenesis Phases

Multiplication

Growth

Maturation

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Chromosome No.

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Types of germ cells

Oogonium (2N)

Additional oogonium (2N)

Primary oocyte (2N)

Secondary oocyte (N) First polar body (N)

Second polar bodies (N)

(All degenerate)

Sperm (N)

Mitosis

Meiosis I

Meiosis II

Types of divisions

Zygote (2N)

Nuclei fuse

Mature ovum (N)

Events

Before birth: Takes place in fetal ovary

After puberty: occurs each month in ovary

Ovulation

Fertilization

Occurs as a result of fertilization

Male pronucleus

Female pronucleus

Note: Egg completes meiosis II after fertilization and second polar bodies present

Before puberty

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Fertilized egg (zygote)

Unfertilized egg

- Ovulation when LH surge (~day 14 of

1 menstrual cycle, 28 days)

- Corpus luteum formed after ovulation

- Progesterone production from corpus

luteum

- No fertilization, corpus luteum degenerated to be corpus albicans

LH surge Ovulation Corpus luteum Progesterone Triggers Forms Produces

High level of progesterone found at luteal phase of ovary

FOLLICULAR PHASE

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Resource: https://ars.els-cdn.com/content/image/1-s2.0-S0011224015002242-gr1.jpg

ZP3

- The receptor on the ZP - Binds sperm at the beginning of fertilization process

The human zona pellucida (ZP) is

composed of three major glycoproteins: ZP1, ZP2, and ZP3.

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Approximate number of oocytes in female RP life time

Primary oocytes

Secondary oocytes

At 5 months of fetal life ~ 7 million

At birth ~ 2 million (5 million degenerated)

At puberty ~ 40,000 cells (1.96 million more generated)

•12 secondary oocytes are ovulated per year

• Up to 480 oocytes over the entire reproductive life of the woman

(40 years x 12 oocytes per year = 480 oocytes )

Ovulation Cumulus oocyte complex (COC)

MII oocyte viability after ovulation for 72 hours

Ovulated oocyte will be successfully fertilized within 12-24 hrs.

If no fertilization occurs, oocyte will degenerate within the oviduct.

Ovulation: LH surge

Ovulated cumulus oocyte complex (COC)

48 Resource: http://bioserv.fiu.edu/~walterm/GenBio2004/new_reprostuff2/chapter_44.htm

Fallopian tube (oviduct)

- Internal fertilization takes place at 1/3 anterior or ampulla part - Gametes and zygote transport

Uterus

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Capacitation of sperm in oviduct

- Reduction in membrane cholesterols

- Hyperactiation: lateral movements

- Removal of coating factors

Note: only capacitated sperm

can bind to the ZP

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Comparisons between spermatogenesis & oogenesis

Outline of the fertilization process

Fertilization

Requirements: 2 steps of sperm

1. Sperm capacitation 2. Sperm acrosome reaction

7 steps of human fertilization

From: Yu’s thesis, 2008 (Queen’s university)

Histologic sperm acrosome reaction

Intact

Intermediate

Reacted

ZP3-INDUCED ACROSOME REACTION

“Supramicromolar calcium influx is essential for the induction of the

acrosome reaction”

Yanagimachi, 2009

Egg activation

“Supramicromolar calcium influx is also essential

egg activation”

Fertilization and implantation