fisiologi reproduksi.

PRINSIP FISIOLOGIS
BERBAGAI MACAM
ESAI HORMON
Kuliah 4
Rahmatina B. Herman
Bagian Fisiologi
Fakultas Kedokteran Universitas Andalas

Hormones
1. Hormones are chemical messengers that
enter the blood, which carries them from
endocrine glands to the target cells

2. Hormone action in the target cells/organ
3. Hormone metabolism

4. Hormone removal or clearance

Hormone Concentrations in Blood
Most hormones are present in blood in
extremely minute quantities, some in

concentrations of pg / ml (1 pg = 1 billionth of
mg)

Almost impossible to measure by usual
chemical means
Extremely sensitive methods:
radioimmunoassay

Hormones Removal From Plasma
Hormones are cleared from plasma in several
ways:
1. Metabolic destruction by tissues
2. Binding with tissues
3. Excretion by liver into bile
4. Excretion by kidneys into urine
A decreased Metabolic Clearance Rate of
hormone causes excessively high concentration
of the hormone in body fluids

Metabolic Clearance Rate of Hormones

Two factors can affect hormone concentration
in blood:
- rate of secretion
- rate of removal which is called metabolic
clearance rate
This is expressed in terms of the number of
ml of plasma cleared of the hormone per
minute

Hormones Measurement
1. Measurement of hormone secretion rate

2. Measurement of circulating hormones
concentration
3. Measurement of hormone action
4. Measurement of hormone metabolite
product
5. Measurement of hormone excretion

Measurement of Hormone Secretion Rate

A simple method for estimating hormone
secretion is:
- Measuring the concentration of natural
hormone in plasma by means of a
radioimmunoassay procedure (C)
- Measuring metabolic clearance rate (MCR)

- By multiplying C x MCR, one derives a value
that is equal to steady-state of hormone
production/ hormone secretion

Measure e t of Hor o e Se retio Rate.….
However, hormone production often increases or
decreases rapidly
In such case, one can measure the changing rate of
secretions only by:
- collecting samples of arterial blood entering the
gland (AB) and samples of venous blood leaving the
gland (VB)
- measuring rate blood flow through the gland (BF)

- by multiplying BF x (VB-AB), one can derive the
instantaneous secretion rate

Measurement of Hormones Concentration
Physiologically variable that fluctuates
each day with a cyclical periodically
Measurements of particular variable are
usually obtained at a single time of day
In certain types of hormonal diseases,
plasma concentration of the hormone
may be normal at one time, but higher at
other time

Measure e t of Hor o es Co e tratio …..
Thus, if the hormone in the blood was measured
at only one time of day, the disorder might be
missed
To avoid this problem is to obtain repeated
measurements of the hormone over a 24-hour
period

Ideally, repeated blood measurements could be
drawn to provide as complete a profile as possible
of the minute-to-minute changes in circulating
hormone level.
But it is not practical in practice

Measurement of Hormones Concentration…..
A simpler method is to obtain a 24-hour
cumulative urine sample
Metabolites of many hormones appear in the
urine as part of the daily process of clearing excess
hormones from the blood
The more hormones in the blood, the more it or its
metabolites appear in the urine
A-24 hour measurement will provide information
on the integrated, or summed, amount of
hormone produced during the day and night.
So that it is a time-averaged mean

Measurement of Hormones Concentration…..

In fact that time-averaged means reveal
nothing of the countless small (sometimes
large) fluctuations in circulating hormone
concentration that occurred during that time
It reveals whether or not abnormally low or
high total amounts of hormone were produced

Measurement of Hormones Through Its Action

Different hormone has different effects on
target organ, so that the effects of the
hormones on the target organ may reflect
the hormone secretion or production
For examples: Basal Metabolic Rate (BMR)
may reflect thyroid hormones

Measure e t of hor o es through its a tio …..

Hormones has pharmacological effects on
target organs so that it also may reflect

the concentration of that hormone

For examples: measurement of
cardiovascular parameters may reflect
sympathetic neurohormones

Measurements of
Metabolic Clearance Rate of Hormones
To calculate Metabolic Clearance Rate (MRC),
one makes following 2 measurements:
1. Rate of disappearance of the hormone
from plasma per minute (D)
2. Concentration of the hormone in each
ml plasma (C)
D
MCR = ---C

How to Measure
Quantitative:
- Blood samples : hormones

- Urine samples : metabolites product
hormone excretion
Qualitative:
- direct effect on target organ
- pharmacological effect

Ho to Measure..…
Blood samples: hormones
Most hormones are unstable, so that need
appropriate approach:
- before assaying
> drawing samples
> transportation: temperature
> storing: temperature and long life
- during assaying:
> direct assessment
> indirect / through appropriate process:
derivatization process

Ho to Measure..…

Valid and reliable
on method, tools, competencies
- Intra-assay validation (intra-day
validation)
- Inter-assay validation (inter-day
validation)

- Standard Curve

Guideline on Bioanalytical
Method Validation
(Committee for Medicinal Products for
Human Use / CHMP, 2011)

Method Validation
The main objective of method validation is to
demonstrate the reliability of a particular method for
the determination of an analyte concentration in a
specific biological matrix, such as blood, serum,
plasma, urine, or saliva

If an anticoagulant is used, validation should be
performed using the same anticoagulant as for the
study samples
Generally a full validation should be performed for
each species and matrix concerned

Method Validatio …..
Main characteristics of bioanalytical method that are
essential to ensure the acceptability of the
performance and the reliability of analytical results are:
- Selectivity
- Lower limit of quantification (LLOQ)
- the response function and calibration range
(calibration curve performance / standard curve)
- Accuracy
- Precision
- Matrix effects
- Stability of the analytes in biological matrix
- Stability of the analytes and of internal standard (IS) in

the stock and working solutions and in extracts under
the entire period of storage and processing conditions

Method Validatio …..
During method validation and analysis of study
sample, a blank biological matrix will be spiked
with the analytes of interest using solutions of
reference standards to prepare calibration,
standards quality control samples and stability
samples
In addition, suitable internal standards (IS) can
be added during sample processing in
chromatographic method

Selectivity
The analytical method should be able to differentiate
the analytes of interest and internal standard (IS) from
endogenous components in the matrix or other
component in the sample
Selectivity should be proved using at least 6 individual
sources of the appropriate blank matrix, which are
individually analysed and evaluated for interference
Normally, absence of interfering components is
accepted where the response is < 20% of the lower
limit of quantification for the analyte and 5% for the
internal standard (IS)

Lower Limit of Quantification (LLOQ)
LLOQ is the lowest concentration in a sample which
can be quantified reliably, with an acceptable accuracy
LLOQ is considered being the lowest calibration
standard
The analyte signal of LLOQ sample should be at least 5
times the signal of blank sample
LLOQ should be adapted to expected concentrations
and to the aim of study: for bioequivalence studies
LLOQ should be not higher than 5% of Cmax
LLOQ may be not necessary for exploratory
pharmacokinetic studies

Calibration Curve Performance
Before carrying out the validation of the analytical
method, it should be known what concentration range is
expected
The range should be covered by calibration curve range,
defined by LLOQ being the lowest calibration standard and
the upper limit of quantification (ULOQ) being the highest
calibration standard
A minimum of 6 calibration concentration levels should be
used, in addition to the blank sample (processed matrix
sample without analyte and without IS) and a zero sample
(processed matrix with IS)
Each calibration standard can be analysed in replicate

Cali ratio Curve Perfor a e…..
The calibration curve parameters should be
reported (slope and intercept in case of linear fit)
The back calculated concentrations of the
calibration standards should be presented
together with the calculated mean accuracy values
All the available (or acceptable) curves obtained
during validation, with a minimum of a 3 should
be reported

Cali ratio Curve Perfor a e…..
The back calculated concentrations should be within
±15% of the nominal value, except for LLOQ for which
it should be within ±20%
At least 75% of calibration standards, with a minimum
of 6 calibration standard levels, must fulfill this
criterion
In case replicates are used, the criteria (within ±15% or
±20% for LLOQ) should also be fulfilled for at least 50%
of calibration standards tested per concentration level
In case a calibration standard does not comply with
these criteria, this calibration standard sample should
be rejected

Standard Curve
A standard curve is a type of graph used as a
quantitative research technique.
A graphic plot of tracer binding versus the known
concentration of test substances in a set of standards
usually prepared by serial dilution or incremental
addition.
Multiple samples with known properties are
measured and graphed

So then allows the same properties to be determined
for unknown samples by interpolation on the graph.

Accuracy
Within-run accuracy
Determined by analysing in a single run a minimum of 5
samples per level at a minimum 4 concentration levels
which are covering the calibration range.
The mean concentration should be within 15% of nominal
values, except for LLOQ should be 20% of the nominal value

Between-run accuracy
For the between-run accuracy, LLOQ, low, medium and high
QC samples from at least 3 runs analysed on at least 2
different days should be evaluated
The mean concentration should be within 15% of the
nominal values for the QC samples, except for LLOQ which
should be within 20% of the nominal value)

Precision
Within-run precision
For the validation of the within-run precision, there should
be a minimum of 5 samples per concentration level at
LLOQ, low, medium and high QC samples in a single run
The within-run CV value should not exceed 15% for QC
samples, 20% for LLOQ

Between-run precision
For the validation of the between-in run precision, LLOQ,
low, medium and high samples from at least 3 runs
analysed on at least 2 different days should be evaluated
The between-run CV value should not exceed 15% for QC
samples, 20% for LLOQ

Matrix Effects
Matrix effects should be investigated when using mass
spectrometric methods
Using at least 6 lots of blank matrix from individual donors
For each analyte and IS, matrix factor should be calculated
for each lot of matrix, by calculating the ratio of peak area
in the presence of matrix (measured by analysing blank
matrix spiked after extraction with analyte), to the peak
area in absence of matrix (pure solution of analyte)
The IS normalized MF should also be calculated by dividing
the MF of analyte by the MF of the IS
The CV of IS normalized MF calculated from 6 lots of matrix
should not be greater than 15%
This determination should be done at a low and at a high
level of concentration (maximum of 3 times LLOQ and
close to ULOQ)

Stability
Evaluation of stability should be carried out to ensure that
every step taken during sample preparation and sample
analysis, as well as the storage conditions used do not
affect the concentration of analyte
The following stability tests should be evaluated:
- stability of the stock solution and working solutions of
the analyte and internal standard (IS)
- freeze and thaw stability of the analyte in the matrix
from freezer storage conditions to room temperature or
sampling processing temperature
- short term stability of the analyte in matrix at room
temperature or sampling processing temperature
- long term stability of analyte in matrix stored in freezer
- on-instrument/ autosampler stability of the processed sample
at injector or autosampler temperature

Percentage recoveries of internal standard amitriptyline
Concentration
of amitriptyline

20000pg/ml

Pure AT

Extracted AT

Recovery

(height in chrom.)

(height in chrom.)

(%)

31485
25358
28832
31115
27719
28899
32143

28922
20594
26866
29189
22865
21839
25846

91.86
81.21
93.18
93.81
82.49
75.57
80.41

Mean  SD: 85.50  7.31

Percentage recoveries of derivatized NE
Concentration
of NE

16000 pg/ml
8000 pg/ml

4000 pg/ml
2000 pg/ml

1000 pg/ml
500 pg/ml
250 pg/ml

Pure NE

Extracted NE

Recovery

(height in chrom.)

(height in chrom.)

(%)

103455
109876
83387
104638
53549
56791
42013
52995

86.10
82.49
83.78
85.00

25711
28903
20876
26894
12894
13764
11143
13547

89075
90632
69863
88943
44232
44345
35294
45046
21698
24346
17745
23675
10521
11230
8971
11693

6690
6902
5621
6854
3112
3487
2876
3269
1615
1775
1313
1656

5429
5885
4525
5713
2471
2959
2386
2879
1265
1473
1089
1334

82.60
78.08
84.01
85.00
84.39
84.23
85.00
88.83
81.59
81.59
80.51
86.31
81.15
85.27
80.50
83.35
85.83
84.86
82.96
88.07
78.33
82.99
82.94
80.56

Mean  SD of all concentrations: 83.44  2.59

Mean  SD
84.341.56
82.423.06

85.411.78
82.502.59

82.572.18
85.432.13
81.202.23

Above: Scanning chromatogram of derivatized extracted NE (DNE) and extracted
amitriptyline (AT)
Below: Selected ion monitoring (SIM) chromatogram of derivatized extracted NE (left);
extracted amitriptyline (right)

Peak ratio of derivatized NE and amitriptyline (for standard curve)
Concentra
tion of NE
(pg/ml)

8000
4000
2000
1000
500
100 (LLOQ)

Peak ratio

Mean  SD

1st run

2nd run

3rd run

4th run

1.59
0.86
0.39
0.21
0.117
0.019

1.64
0.90
0.40
0.22
0.111
0.024

1.72
0.81
0.41
0.19
0.107
0.026

1.67
0.83
0.43
0.20
0.099
0.021

LLOQ, lower limit of quantitation

1.65  0.05
0.85  0.04
0.41  0.02
0.21  0.01
0.11  0.008
0.023  0.003

y = 0 ,0 0 0 2 x
R

1,8

2

= 0 ,9 9 9 7

1,6

Peak Ratio

1,4
1,2
1
0,8
0,6
0,4
0,2
0
0

1000

2000

3000

4000

5000

6000

7000

8000

9000

C o ncent rat io n o f N o repinephrine ( pg/ m l)

The standard curve based on peak ratio of derivatized NE and internal
standard amitriptyline. The concentrations of NE were from 8000 pg/ml
to 500 pg/ml and 100 pg/ml, the lower limit of quantitation (LLOQ).

Accuracy and precision of the assay for NE with amitriptyline as internal
standard (n=4)

Conc
of
NE

8000
4000
2000
1000
500
100

Intra-assay variation
(Intra-day variation)

Inter-assay variation
(Inter-day variation)

Peak ratio

Peak ratio
Mean  SD

1st
run

2nd
run

3rd
run

4th
run

1.67
0.90
0.41
0.22
0.121
0.024

1.62
0.86
0.42
0.20
0.118
0.028

1.70
0.82
0.44
0.19
0.129
0.026

1.65
0.88
0.40
0.20
0.124
0.021

1.66  0.03
0.87  0.03
0.42  0.02
0.20  0.01
0.123  0.005
0.025  0.003

CV
(%)

1.81
3.45
4.76
5.00
4.07
12.00

Mean  SD

1st
run

2nd
run

3rd
run

4th
run

1.67
0.90
0.41
0.22
0.121
0.024

1.59
0.82
0.40
0.20
0.117
0.026

1.68
0.86
0.43
0.19
0.119
0.027

1.72
0.83
0.44
0.21
0.127
0.020

1.660.05
0.850.04
0.420.02
0.210.01
0.1210.004
0.024  0.03

(LLOQ)

CV, Coefficient of variation; LLOQ, lower limit of quantitation; Conc,
concentration

CV
(%)

3.01
4.71
4.76
4.76
3.31
12.50

Radioimmuno-Assay

Radioimmuno-Assay
For hormones assessment
Principle of radioimmuno-assay:
Antibody (globulin) which is specific for the hormone
that will be assessed must be produced from the
animal in a great amount (commercially available)
The antibody then mixed with:
- animal serum which contain hormone to be
assessed (h)
- pure standard hormone which has been labeled
by radioisotop (hsr) (with known amount)

Radioimmuno-Assay..…
Antibody and hormone will be bound (ab-h &
ab-hsr)
Hormone to be assessed and hormone labeled
by radioisotope competitively binds the
antibody
Concentration of ab-hsr then measured, soon
after the binding has reached equilibrium,
using radioactive-counting technique

Radioimmuno-Assay..…
To make assay highly ua titative ,
radioimuno-assay must also be applied for
sta dard solution from pure un-labeled
hormone with some levels of
concentration
The results then will be arranged in a
Standard Curve

PENGANTAR
FISIOLOGI REPRODUKSI
Kuliah 1
Rahmatina B. Herman
Bagian Fisiologi
Fakultas Kedokteran Universitas Andalas

Reproduction
Reproduction is process to maintain
continuation of species by which
- new individuals of a species are produced
- genetic material is passed from generation
to generation
Cell division in a multicellular organism is
necessary for growth and it involves passing of
genetic material from parent cells to daughter
cells
Performed by reproductive system

The Reproductive System
does not contribute to homeostasis

is not essential for survival of an individual
But still plays a i porta t i a perso ’s life,
e.g. the manner:
- in which people relate as sexual beings
contributes in significant ways to
psychosocial behavior
- how people view themselves
- how people interact with others

The Reprodu tive Syste …..
Reproductive function also has a profound
effect on society:
- universal organization of societies into
family units provide a stable environment
that is conducive for perpetuating our
species
- on other hand, population explosion and its
resultant drain on dwindling resources
have led to worldwide concern with means
by which reproduction can be limited

The Reprodu tive Syste …..
Reproductive capability depends on intricate
relationship among hypothalamus, anterior
pituitary, reproductive organs, and target cells
of sex hormones

These relationship employ many of regulatory
mechanisms used by other body systems for
maintaining homeostasis, such as negativefeedback control

The Reprodu tive Syste …..
Sexual behavior and attitudes are deeply
influenced by emotional factors and sociocultural mores of the society in which the
individual lives

However, Reproductive Physiology will
concentrate on basic sexual and reproductive
functions that are under nervous and hormonal
control, and will not examine physiological and
social ramifications of sexual behavior

The Reprodu tive Syste …..
The organ of male and female may be grouped
by function
Testes and ovaries (gonads), function in
production of gametes: sperm and ova

Gonads also secrete hormones
The ducts of reproductive systems transport,
receive, and store gametes
Accessory sex glands produce materials that
support gametes

The Reproductive System…..
In females, the breasts are also considered
accessory reproductive organs
The externally visible portions of reproductive
system are known as external genitalia

The production of gametes and fluid, and their
discharge into ducts classify the gonads as
exocrine glands
Whereas the production of hormones classify
the gonads as endocrine glands

Secondary Sexual Characteristic
Secondary sexual characteristic are many external
characteristics not directly involved in
reproduction
That distinguish male and female

Development and maintenance governed by
testosterone in males and estrogen in females
Progesterone has no influence on secondary
sexual characteristic
Axillary and pubic hair growth is not secondary
sexual characteristic

Secondary Sexual Characteristic..…
In some species, secondary sexual
characteristic are great importance in courting
a d ati g ehavior e.g. to attra t fe ale’s
attention)

In humans, attraction the opposite sex not only
influenced by secondary sexual characteristic
but also strongly affected by the complexities
of human society and cultural behavior

Overview of Functions and Organs
of Male Reproductive System
The essential reproductive functions of male are:
1. Production of sperm (spermatogenesis) by
testes (in skin-covered sac: scrotum)
2. Delivery of sperm to female – semen by
- male reproductive tract: epididymis, vas
deferens, ejaculatory duct
- urethra (in penis)
3. Male accessory sex glands: providing bulk of
semen: seminal vesicle, prostate,
bulbourethral gland

Overview of Functions and Organs
of Female Reproductive System
Female’s role i reprodu tio is

ore o pli ated:

1. Production of ova (oogenesis) by ovaries
2. Reception of sperm: vagina-cervix
3. Reception of sperm and ovum to a common site for
union (fertilization or conception): Fallopian tube
4. Maintenance of the developing fetus until it can
survive in outside world (gestation or pregnancy),
including formation of placenta (organ exchange
between mother and fetus): uterus
5. Giving birth to the baby (parturition)
6. Nourishing the infant after birth by milk production
(lactation): mammae

Overview of Functions and Organs
of Fe ale Reprodu tive Syste …..
Product of fertilization: embryo
During first 2 months of intrauterine
development when tissue differentiation is
taking place

Developing living being is recognizable as
human: fetus
- no further tissue differentiation
- tremendous tissue growth and maturation

Overview of Functions and Organs
of Fe ale Reprodu tive Syste …..
Female reproductive tract consists of:
Ovaries
Oviduct s (Fallopian tubes)
- pick up ova on ovulation and serve as fertilization site

Uterus, thick-walled hollow: responsible for
- maintaining fetus during development , and
- expelling it at the end of pregnancy

Cervical canal
- small opening of cervix
- pathway for sperm to uterus then to oviduct
- passageway for delivery of baby from uterus

Cervix
- lowest portion of uterus which projects into vagina

Overview of Functions and Organs
of Fe ale Reprodu tive Syste ……
Vagina
- expandable tube
- connects uterus to external environment

Vaginal opening
- located in perineal region
- between urethral opening and anal opening

Hymen
- thin mucus membrane partially covering vaginal opening

• Labia minora and labia majora
- skin folds surrounding vaginal and urethral openings

• Clitoris
Female external genitalia collectively: vulva

Sex Determination and Differentiation
Reproductive cells each contain a half set of
chromosomes
Gametogenesis is accomplished by meiosis
The sex of and individual is determined by
combination of sex chromosomes
Sexual differentiation along male or female
lines depends on the presence/ absence of
masculinizing determinant

Parents with diploid (46 chr) somatic cells

Mother

Father
Meiotic division
of germ cells

Meiotic division
of germ cells

Haploid Sperm

Haploid Ovum
Fertilization

Diploid fertilized Ovum
Mitosis

Offspring of diploid somatic cells

Ovum with X sex chromosome
Sperm with Y sc

Embryo with XY sc

Fertilized by

Genetic sex

Embryo with XX sc

No Y chr, so no SRY
and no H-Y antigen

Sex-determining region
of Y chr (SRY) stimulates
Production of H-Y antigen
In plasma membrane of
undifferentiated gonad
H-Y antigen directs
differentiation
of gonads into testes

Sperm with X sc

Gonadal
sex

With no H-Y antigen,
undifferentiated gonads
develop into ovaries

Testes secrete hormone and factor
Testosterone

Mullerian-inhibiting factor

Converted to
Degeneration of
Mullerian ducts

Dihydrotestosterone
(DHT)
Promotes development of
undifferentiated external
genitalia along male lines
(e.g. penis, scrotum)

Phenotype
sex

Transforms Wolfian ducts
into male reproductive tract
(e.g. epididymis, ductus
deferens, ejaculatory duct,
seminal vesicle)

Ovaries does not secrete hormone and factor
Absence of testosterone

Absence of Mullerianinhibiting factor

Degeneration of
Wolfian ducts
Undifferentiated external
genitalia along female lines
(e.g. clitoris. labia)
Phenotype
sex

Mullerian ducts develop
Into female reproductive
tract (e.g. oviducts, uterus)

Errors in Sexual Differentiation
Genetic sex and phenotype sex are usually
compatible
Occasionally, discrepancies occur
between genetic and anatomic sexes
because of errors in sexual differentiation

Errors i Se ual Differe tiatio …..
1. If testes in a genetic male fail to properly
differentiate and secrete hormones, the result
is the development of an apparent anatomic
female in a genetic male, who, of course will
be sterile.
Similarly, genetic males whose target cells lack
receptors for testosterone are feminized, even
though their testes secrete testosterone

Errors i Se ual Differe tiatio …..
2. Testosterone acts on Wolfian ducts to convert
them into a male reproductive tract;
If testosterone derivative dihydrotestosterone
(DHT) that responsible for masculinization of
external genitalia because of genetic deficiency
of the enzyme which converts testosterone
into DHT, results in a genetic male with testes
and a male reproductive tract but with female
external genitalia

Errors i Se ual Differe tiatio …..
3. Adrenal gland normally secretes a weak
androgen, dehydroepiandrosterone in
insufficient quantities to masculinize females.
If, pathologically excessive secretion of this
hormone in a genetically female fetus during
critical developmental stages imposes
differentiation of reproductive tract and
genitalia along males lines

Errors i Se ual Differe tiatio …..
Sometimes, the discrepancies between genetic
sex and apparent sex are not recognized until
puberty, when discovery produces
psychologically traumatic gender identity crisis

For instance: a masculinized genetic female
with ovaries, but with male type external
genitalia may be reared as a boy until puberty.
When breast enlargement and lack of beard
growth signal an apparent problem

Errors i Se ual Differe tiatio …….
Less dramatic cases of inappropriate sex
differentiation often appear as sterility
problems
Therefore, important to diagnose any
problems in sexual differentiation in infancy. It
can be reinforced, if necessary, with surgical
and hormonal treatment, so that psychosexual
development can proceed as normally as
possible

Tugas
1. Hubungan sistem limbik (limbic system)
dengan pengaturan fungsi seks

2. Hubungan kelenjar pineal (pineal body)
dengan pegaturan fungsi seks

DASAR-DASAR
BIOMOLEKULER
REPRODUKSI WANITA
Kuliah 2
Rahmatina B. Herman
Bagian Fisiologi
Fakultas Kedokteran Universitas Andalas

Sex Determination and Differentiation
Reproductive cells/gamete each contain a half
set of chromosomes (haploid)
Gametogenesis is accomplished by meiosis
The sex of and individual is determined by
combination of sex chromosomes
Sexual differentiation along male or female
lines depends on the presence/absence of
masculinizing determinant

Ovaries does not secrete hormone and factor

Absence of testosterone

Absence of Mullerianinhibiting factor

Degeneration of
Wolfian ducts

Undifferentiated external
genitalia along female lines
(e.g. clitoris. labia)

Phenotype
sex

Mullerian ducts develop
Into female reproductive
tract (e.g. oviducts, uterus)

/Ovarian agenesis/Turner syndrome

/Klinefelter syndrome

Summary of 4 possible defects produced by maternal nondisjunction of sex chromosomes
at the time of meiosis
The YO combination is believed to be lethal, the fetus dies in utero

Female Reproductive System
Reproductive system of women shows regular cyclic
changes that may be regarded as periodic preparation for
fertilization and pregnancy
In humans and primate, the cycle is a menstrual cycle and
its conspicuous feature is periodic vaginal bleeding that
occurs with the shedding of uterine mucosa (menstruation)
In other mammal: the sexual cycle is called estrous cycle,
no episodic vaginal bleeding occurs, but the underlying
endocrine events are essentially similar
- in some species: ovulation occurs spontaneously
- in other species: ovulation is induced by copulation
(reflex ovulation)

Ovaries
Primary female reproductive organs

Perform dual function:
- producing ova (oogenesis)
- secreting female sex hormones:
estrogen and progesterone which act together to:
> promote fertilization of ovum
> prepare female reproductive system for pregnancy

Containing various levels of follicle
development

Histology of the ovary. The arrows indicate the sequence of developmental stages
that occur as part of ovarian cycle

Oogenesis
Undergo numerous mitotic divisions

± 7 month after conception, fetal oogonia
cease dividing
From this point on, no new germ cells are
generated
Still in the fetus, all oogonia develop into
primary oocytes

Primary Oocyte
Begin a first meiotic division by replicating their DNA
However, they do not complete the division in the
fetus
Accordingly, all the eggs present at birth are primary
oocytes containing 46 chromosomes, each with two
sister chromatids
Cells are said to be in a state meiotic arrest
State meiotic arrest continues until puberty and the
onset of renewed activity in ovaries
Only primary oocytes destined for ovulation will ever
complete the first meiotic division, for it occurs just
before the egg is ovulated

Pri ary Oo yte…..
Each daughter cells receives 23 chromosomes, each
with 2 chromatids
One of the two daughter cells, secondary oocytes
retains virtually all cytoplasm (other is first polar
body)

Thus, the primary oocytes:
- Already as large as the egg will be
- Passes on to be secondary oocyte half of its
chromosomes but almost all of its nutrient-rich
cytoplasm

Secondary Oocyte
The second meiotic division occurs in a
fallopian tube after ovulation, but only if the
secondary oocyte is fertilized (penetrated by a
sperm)

Daughter cells each receive 23 chromosomes,
each with a single chromatid
One of the two daughter cells, termed an ovum
retains nearly all cytoplasm (other is second
polar body)

Final Result of Oogenesis

Net result of oogenesis is
that each primary oocyte
can produce only one
ovum

Oogenesis. 2n means diploid (46 chromosomes; n means haploid (23 chromosomes)

Summary of Oogenesis
Oogonia

Chromosomes
Per cell

Chromatids
Per cell

46

2

46

2

23

2

23

1

Birth

Puberty

Summary of Ooge esis…..
Oogonia: mitotic divisions until ± 7 month after
conception

Mitosis of oogo iu

→ primary oocyte

Meiosis of primary oocyte, but do not complete
(beginning of the 1st eioti divisio → eioti arrest

Primary oocyte at birth containing 46 chromosomes
1st meiotic division is completed just before ovulation
→ secondary oocyte

2nd meiotic division occurs in a fallopian tube after
ovulation, but only if the secondary oocyte is fertilized
(penetrated by a sperm)

Comparison of Spermatogenesis and Oogenesis

Spermatogenesis
Three major stages:
1. Mitotic proliferation
2. Meiosis
3. Packaging/ spermiogenesis: physically
reshaping/ remodeling
± 64 days, from spermatogonium to mature sperm
Up to several hundred million sperm may reach
maturity daily

Follicle
From the time of birth, there are many primordial
follicles, each containing 1 primary oocyte
Progression of some primordial follicles to
preantral and early antral stages occurs
- throughout infancy and childhood, and
- then during the entire menstrual cycle
Therefore, although most of follicles in ovaries are
still primordial, there are also always present a
relatively constant few number of preantral and
early antral follicles

Menstrual Cycle
At the start of each menstrual cycle, 10-25 the

follicles begin to develop into larger follicles
In humans, usually one of the larger follicles in one
ovary starts to grow rapidly on ± the 6thday, becomes
the dominant follicle

The dominant follicle continues to develop, and
others (in both ovaries) regress and become a
degenerative process called atresia (an example of
programmed cell death, or apoptosis)
The eggs in the degenerating follicles also die

Ovulation
Mature follicle (Graafian follicle): ± 1,5 cm in
dia eter, that it alloo s out o ovary’s surfa e
Ovulation occurs when the thin walls of follicle
and ovary at site where they are joined rupture
because of enzymatic digestion
Secondary oocyte surrounded by its tightly
adhering zona pellucida and granulosa cells, as
well as cumulus, is carried out of ovary and onto
ovarian surface by antral fluid

Ovulatio …..
Occasionally, 2 or more follicles reach
maturity and more than 1 egg may be
ovulated
This is the most common of cause of
multiple births
In such cases, siblings are fraternal, not
identical, because the eggs carry different
sets of genes

Postulated mechanism of ovulation (H. Lipner)

LH
Follicular steroid hormones (progesterone)
Proteolytic enzymes
(collagenase)

Follicular hyperemia
and
Prostaglandin secretion

Weakened follicle wall

Plasma transudation
into follicle

Degeneration
of stigma

Follicle swelling

Follicle rupture

Evagination of ovum

Positive feedback of high levels of estrogens on secretion of GnRH and LH

Indicators of Ovulation
A surge in LH secretion triggers ovulation
- Ovulation normally occurs ± 9 h after the peak of LH surge
- The ovum lives for ± 72 h after ovulation, but it is
fertilizable for a much shorter time

Research shows:
>
>
>
>

Intercourse on the day of ovulation: pregnancy 36%
Intercourse on days after ovulation: pregnancy 0
Intercourse 1-2 d before ovulation: pregnancy 36%
A few pregnancies resulted from intercourse 3-5 d before
ovulation (8% on day 5 before ovulation)

- Thus, some sperms can survive in the female genital tract
and fertilize the ovum for up to 120 h before ovulation,
but the most fertile period is clearly 48 h before ovulation

I di ators of Ovulatio …..
A change (usually rise) in basal body temperature
caused by secretion of progesterone, since
progesterone is thermogenic
- The rise starts 1-2 d after ovulation
- Obtaining an accurate temperature chart should
use a digital thermometer and take oral/rectal
temperatures in the morning before getting out
of bed
- Temperature change at the time of ovulation is
probably caused by the increase in progesterone
secretion

Basal body temperature and plasma LH and FSH concentrations (mean ± SE)
during the normal human menstrual cycle

Formation of Corpus Luteum
After mature follicle discharges its antral fluid and
egg, it collapses around antrum and undergoes rapid
transformation
Granulosa cells enlarge greatly, and entire glandlike
structure formed, known as corpus luteum (CL)

CL secretes estrogen, progesterone, inhibin
If the discharged egg (now in a fallopian tube) is not
fertilized, CL reaches its maximum development
within ± 10 days.
CL then rapidly degenerates by apoptosis
It leads to menstruation and beginning of a new
menstrual cycle

Granulosa Cell
Primordial follicles surrounded by a single layer of
granulosa cells
Granulosa cells secrete:
- estrogen ,
- small amounts of progesterone just before
ovulation
- peptide hormone inhibin
During childhood granulosa cells secrete:
- nourishment for ova
- maturation inhibiting factor

Gra ulosa Cell…..
Further development from primordial follicle stage is
characterized by
- an increase in size of oocyte
- a proliferation of granulosa cells into multiple layers
- separation of oocyte from inner granulosa cells by a
thick of material: zona pellucida
Granulosa cells produce one or more factors that act
on primary oocytes to maintain them in meiotic arrest

Gra ulosa Cell…..
Inner layer of granulosa cells remains closely
associated with oocyte by means of
cytoplasmic processes that traverse zona
pellucida and form gap junctions with oocyte

Nutrients and chemical messengers are passed
to oocyte through gap junctions
Granulosa cells produce one or more factors
that act on primary oocytes to maintain them
in meiotic arrest

Theca Formation
As follicle grows by mitosis of granulosa cells,
connective tissue cells surrounding granulosa
cells differentiate and form layers known as
theca

Shortly after theca formation,
- Primary oocyte reaches full size (115 m in
diameter)
- Antrum (fluid-filled space) begins to for in
the midst of granulosa cells as result of fluid
they secrete

Illustration of an ovary shows sequential development of follicle, the formation of
corpus luteum and follicular atresia

Process of Atresia
Atresia is not limited to just antral follicles, follicles
can undergo atresia at any stage
This process is already occurring in utero so that
the 2-4 million follicles and eggs are present at
birth represent only a small fraction of those
present at earlier time in the fetus
Atresia then continues all through pubertal life so
that only 200,000-400,000 follicles remain when
active reproductive life begins.
Atresia still continues during reproductive life

Pro ess of Atresia…..
Therefore, 99,99 % of ovarian
follicles present at birth will
undergo atresia

Sites of Synthesis of Ovarian Hormone
Estrogen is synthesized and released into blood:
- during follicular phase mainly by granulosa cells
- after ovulation, by CL
Progesterone is synthesized and released into
blood:
- in very small amounts by granulosa and theca
cells just before ovulation
- major source is CL (after ovulation)
Inhibin is synthesized and released into blood:
- by granulosa cells
- CL

Interactions between theca and granulosa cells in estradiol
synthesis and secretion

Ovarian Cycle
1. The follicular phase:
- ovarian follicle growth
- ovulation

2. The luteal phase:
- development of corpus luteum

Uterine Cycle
- estrogen  estrogen phase
- before ovulation

1. Proliferative phase:

2. Secretory phase:

- progesterone  progestational phase
- after ovulation

3. Menstruation
- estrogen & progesterone decreased

Relative concentrations of anterior pituitary gland hormones (FSH – LH) and ovarian
Hormones (estrogen – progesterone) during a normal female sexual cycle. Note
the relationship of the hormones to the ovarian and uterine cycles

Cyclical Changes in Cervix
The mucosa of cervix does not undergo cyclical
desquamation
There are regular changes in cervical mucus:
- Estrogen makes the mucus thinner, watery, and more
alkali e → pro otes the survival a d tra sport of sper s
- At the time of ovulation the mucus is:
> thinnest and fern-like pattern on slide
> its elasti ity i reases → a drop a e stret hed i to a
long (8 - ≥
, a d thi thread
- Progesterone makes the mucus thick, tenacious, and
cellular
- After ovulation and during pregnancy: thick, no fernpattern

Estrogen:
fern-like pattern

Progesterone:
no fern-like pattern

Estrogen,
no progesterone:
Fern-like pattern

Microscopic of patterns formed of cervical mucus on dried smeared slide.
Progesterone makes the mucus thick and cellular.
In anovulatory, no progesterone is present to inhibit fern-like pattern

Cyclical Changes in Vagina
Under influence of estrogen:
- the vaginal epithelium becomes cornified that
can be identified in the vaginal smear
Under influence of progesterone:
- secretion of thick mucus
- the vaginal epithelium proliferates and
becomes infiltrated with leukocytes

The cyclical changes in vaginal smear in rats are
relatively marked; in humans and other species
are similar, but not so clear cut

Cyclical Changes in Breast
Although lactation normally does not occur until the
end of pregnancy, cyclical changes take place in the
breasts during the menstrual cycle
Estrogens cause proliferation of mammary ducts
Progesterone causes: growth of lobules and alveoli
The breast swelling, tenderness, and pain experienced
by many women during the 10 day preceding
e struatio ← due to diste tio of the du ts,
hyperemia, and edema of the breast interstitial tissue
All the changes regress along with symptoms, during
menstruation

Normal Menstruation
Menstrual blood is predominantly arterial, only 25% of
the blood being of venous origin
Containing tissue debris, prostaglandins, and relatively
large amount of fibrinolysin from endometrial tissue
Fi ri olysi lyses lots → o lots i
e strual lood
Usual duration is 3-5 d, but 1-8 d can occur normally
The average amount of blood lost is 30 ml (range
normally from light spotting – 80 ml)
The amount of blood affected by various factors,
including the thickness of endometrium, medication,
and diseases that affect clotting mechanism

Anovulatory Cycles
Anovulatory cycles are common for the first 12-18
months after menarche and before the onset of
menopause
Whe ovulatio does ot o ur → o CL → effe ts of
progesterone on endometrium are absent
Estrogens continue to cause growth, and proliferative
endometrium becomes thick enough to break down
and begins to slough
The time it takes for bleeding usually < 28 d from the
last menstrual period
The flow is variable from scanty to relatively profuse

PERKEMBANGAN
ORGAN REPRODUKSI WANITA
DARI JANIN SAMPAI LAHIR
Kuliah 5
Rahmatina B. Herman
Bagian Fisiologi
Fakultas Kedokteran Universitas Andalas

Overview of The Reproductive System
The organ of male and female may be grouped
by function
Testes and ovaries (called gonads), function in
production of gametes: sperm cells and ova

Gonads also secrete hormones
The ducts of reproductive systems transport,
receive, and store gametes

The externally visible portions of reproductive
system are known as external genitalia

Overview of The Reproductive System…..
Accessory sex glands produce materials that
support gametes
In females, the breasts are also considered
accessory reproductive organs
The production of gametes and fluid, and their
discharge into ducts classify the gonads as exocrine
glands
Whereas the production of hormones classify the
gonads as endocrine glands

Overview of Female Reproductive System
Fe ale’s role i reprodu tio is ore o pli ated tha
ale’s:
1. Production of ova (oogenesis) by ovaries
2. Reception of sperm: vagina-cervix
3. Reception of sperm and ovum to a common site for union
(fertilization or conception): Fallopian tube
4. Maintenance of the developing fetus until it can survive in
outside world (gestation or pregnancy), including
formation of placenta (organ exchange between mother
and fetus): uterus
5. Giving birth to the baby (parturition)
6. Nourishing the infant after birth by milk production
(lactation): mammae

Overview of Female Reproductive Syste …..
Product of fertilization: embryo
During first 2 months of intrauterine development
when tissue differentiation is taking place
Developing living being is recognizable as human:
fetus
- no further tissue differentiation, only
tremendous tissue growth and maturation

Chronology of Reproductive Function
Sequential changes in reproductive development or
function:
Sex determination
Genetic inheritance sets the gender of individual which is
established at the moment of fertilization

Sex differentiation
Multiple process in which development of reproductive system
occurs in fetus

Maturation of system at puberty
Eventual decrease in reproductive function that occurs
with aging

SEX DETERMINATION

Definition
Sex determination is concerned with the regulation of
the development of the primary of gonadal sex, while
sex differentiation encompasses the events
subsequent to gonadal organogenesis
The processes are regulated by at least 70 different
that are located on the sex chromosomes and
autosomes and that act through a variety of
mechanisms including organizing factors, gonadal
steroids and peptide hormones, and tissue receptors
Mammalian embryos remain sexually undifferentiated
until the time of sex determination

Sex Determining Region
An important point: early embryos of both sexes
possess indifferent common primordial that have an
inherent tendency to feminize, unless in the
interference by masculinizing factors
Sex-determining region Y (SRY) gene is found to be the
primary sex determinant that induce the indifferent
gonad into testes
SRY is expressed in XY gonads in Sertoli cell
progenitors at the stage of sex cord formation
In human, SRY gene is located on the short arm of Y
chromosome

Translation of Genetic Sex
The SRY protein is detected at an early age of gonadal
differentiation in XY embryos, where it induces Sertoli cell
differentiation (in adult, it is present in both Sertoli and
germ cells)
In embryonic and fetal life, SRY gene product regulates
gene expression in a cell autonomous manner
The precise molecular mechanisms by which SRY triggers
testis development are unknown, nor is it yet known how
SRY is regulated

The genetic sex of zygote is established by fertilization of a
normal ovum with an X-chromosome by Y-chromosomebearing sperm

Sex Determination

SEX DIFFERENTIATION

Differentiation of Reproductive Tract
Although male and female external genitalia develop
from the same embryonic tissue, but reproductive
tract develop from the different system
2 primitive duct systems: Mullerian ducts and Wolfian
ducts develop in all embryos, so that the early embryo
has potential to develop either a male or a female
reproductive tract
Development of reproductive tract is determined by
the presence of 2 hormones secreted by fetal testes:
testosterone and Mullerian-inhibiting factor
In female fetus: reproductive tract develops from
Mullerian ducts and Wolfian ducts degenerate

Differentiation of Gonads
The genes directly determine only whether the
individual will have testes or ovaries
Sex differentiation depends upon the presence or
absence of substance produced by the genetically
gonads, in particular, testes
Difference between male and female exist at 3 levels:
- genetic sex: depends on combination of sex chr
- gonadal sex: determined by genetic sex , the presence of
a Y hr → SRY → H-Y antigen
- phenotype (anatomic) sex: depends on genetically
determined gonadal sex

Differe tiatio of Go ads…..
The reproductive organs are embryogically developed from
the intermediate mesoderm
The male and female gonads derive from an area called
urogenital ridge, a condensation tissue near adrenal gland
The gonad develops a cortex and a medulla
Until the 6th week of uterine life, primordial gonads are
u differe tiated → ide ti al i oth se es
A gene on Y chromosome (SRY gene) is expressed at this
time in urogenital ridge cells and triggers the development
of testes
Absence of Y chromosome, and hence, SRY gene, testes do
not develop, instead, ovaries begin to develop in the same
area at about 11 weeks

Differe tiatio of Go ads…..
In genetic males, during the 7th and 8th weeks, the medulla
develops into a testis, and the cortex regresses
Leydig a d Sertoli ells appear → testostero e a d MIS are
secreted
In genetic females, at about 11 weeks, the cortex develops
into ovary and the medulla regresses
Embryonic ovary does not secrete hormones
Hormonal treatment of the mother has no effect on
gonadal differentiation in humans
Whe the e ryo has fu tio al testes → ale i ter al
and external genitalia develop

Differentiation of Genitalia
So far as its internal duct system and external genitalia
are concerned, fetus is capable of developing into
either gender
Before the functional of fetal gonads, primitive
reproductive tract includes a double genital duct
system: Wolfian ducts and Mullerian ducts and a
common opening for genital ducts and urinary system
to the outside

Differentiation of Genitalia…..
Normally, most of reproductive tract develops from
only one of the duct systems

In female fetus: Mullerian ducts persist and Wolfian
ducts regress
External genitalia and outer part of vagina do not
develop from the duct system, but from other
structures at body surface
Ovaries (unlike testes), do not play a role in the
development processes of genitalia
In other words, female development occurs
automatically unless stopped from doing so by the
presence of factors released from functioning testes

Differentiation of Internal Genitalia
The internal genitalia are bi-potential until gonads
undergo differentiation
In the 7th week of gestation, the embryo has both
male and female internal genitalia
Internal genitalia develop from the different
system
Two primitive duct systems: paramesonephric
ducts (Mullerian duct) and mesonephric ducts
(Wolffian duct) develop in all embryos, so that the
early embryo has potential to develop either a
male or a female reproductive tract

Differe tiatio of I ter al Ge italia…..
Development of internal genitalia is determined
by the presence of 2 hormones secreted by fetal
testes: testosterone and Mullerian-inhibiting
factor
In normal male fetus, the Wolffian duct system
develops into epididymis ducts and vas deferens,
and Mullerian ducts degenerate

In normal female fetus, the Mullerian duct system
develops into uterine tubes (oviducts) and a
uterus, and Wolffian ducts degenerate

Differentiation of External Genitalia
Male and female external genitalia develop from the
same embryonic tissue
Undifferentiated external genitals consist of:
- Genital tubercle which rise to exquisitely sensitive
erotic tissue
- Urethral folds surrounding a urethral groove
- Genital (labioscrotal) swellings
The external genitalia are bi-potential until the 8th
week of gestation
Thereafter, the urogenital slit disappears → male
genitalia form, or the urogenital slit remains open →
female genitalia form

Differe tiatio of E ter al Ge italia…..
In normal female fetus
Genital tubercle
Develop into clitoris without penetration by urethral
opening
Paired urethral folds
Does not fuse and become labia minora
Urogenital slit
Urogenital slit remains open and become vagina
Genital (labioscrotal) swellings
Does not fuse and become labia majora

DEVELOPMENT OF
REPRODUCTIVE ORGAN
AND FUNCTION

Development of Ovaries
Descend to brim of pelvis during third month of
development
During fetal life, the outer surface of ovary is covered
by a germinal epithelium, which embryologically is
derived from epithelium of germinal ridges
As the female fetus develops, cells that give rise to ova
arise from endoderm of yolk sac and migrate to
ovaries during embryonic