Sex determination and

sex differentiation

*Anti mullerian hormone in mammals

*Regulation of sex differentiation in
invertebrates
*Sexual plasticity
*Allelochemicals
*Environmental endocrine disruptors
Definitions of sex differentiation
and sex determination

Sex determination
is a genotype dependent mechanism (according to the
sex chromosomes) whereby an individual acquires the
gonad type characteristic of one gender of the
species .

Sex differentiation
is the process of changes in cells of indifferent gonads
that lead to formation of embryonic testes or fetal
ovaries as well as processes related to the acquisition
of phenotypic sex.
Anti mullerian hormone (AMH)
also called Mellerian inhibiting
substance (MIS) in vertebrates

A glycoprotein secreted by fetal Sertoli cells

 AMH (MIS)

In the absence of the

AMH initiates the AMH the mullerian
regression of the mullerian duct differentiate into
duct during sexual the uterus fallopian
development of the male tubes and part of the
female vagina

Week 6

This irreversible dissolution in

the human fetus occurs by 51
days post ovulation
 AMH properties

AMH is a 140 kDa homodimeric glycoprotein

linked by disulfide bonds

The human gene encoding AMH is located on

chromosome no. 19.

One of the proteins that control the expression of

AMH, by binding to its gene promoter, is male specific
as it is located on the Y chromosome.
Arnold A Berthold (1803-1861)
and sex steroids

In one of the first endocrine

experiments ever recorded,
Professor Arnold A. Berthold of
Gottingen did a series of tests on
roosters in 1849 while he was
curator of the local zoo.
Berthold Endocrine
Experiment

Remove-
replacement-
injection
Only 60 years
later
testosterone was
discovered
Exposure to testosterone
Males Mix
Sex differentiation in
invertebrates
the crustacean story
 The androgenic gland (AG)
• Male specific
• Regulates masculine development and maintenance

5th WL

5th WL

SD
AG
AG
TA SD
TS

AG
gp 2 mm
Modified from: Ventura et al., Endocrinology 2009
The androgenic gland
(AG) in crustaceans

AG

SD
The androgenic gland and
gonad differentiation
Three brothers
dorsal view
 Androgenic gland
affects growth rate
45
Sham operated males ♂
40
Andrectomized males
35
-AG
Normal females ♂
30
Weight (g)

25 ♀

20
15
10
5
0
0 100 200 300 400
Days after Andrectomy
Dorsal View of sex reversed male
showing ovary development
Applied?
 ♂
X

50% 50% AG

WZ ZZ

Neo X ZZ
ZZ

All male

100 %
ZZ
Aflalo et al., Aquaculture 2006
Vietnam
India
Sexual plasticity Hermaphroditism
and intersexuality

Effects of AG removal in crustaceans

 Intersexuality

An individual of a
bisexual species
that has
characteristics
intermediate
between those of a
female and a male
Sexual plasticity in C. quadricarinatus:
the intersex model for AG removal

Male Intersex Female

 Microsurgical removal of the AG
causes a reproductive shift in
intersex

Control intersex AG ablated intersex

Control intersex AG ablated intersex

Plumose setae Simple and plumose setae
Khalaila
Sagi et et
al.,al.,
20021999
AG ablation permits
Justovarian
a reminder…
activation
DDW -AG
♀ArrestedDDW Active ♂ -AG

2mm 2mm 50µm 50µm

Oocyte Oocyte
diameter diameter
(µm) (µm)
305±77 899±139*
* p<0.05, One-way ANOVA followed by Fisher LSD test
Sex shift – maleness regression

DDW -AG
Active ♂

Testis

50µm 50µm

Sperm duct

50µm 50µm
Escalated fight- behavioral
bioassay for AG activity
AG removal decreases masculine
aggressive behavior
that included escalated fighting

1
Proportion of encounters

0.8

0.6

0.4

0. 2

0
Male- Male- Male- Male-Male
Female AG ablated Intersex
intersex Barki et al., 2006
AG ablated intersexual behaves
as female
AG effects in
crustaceans

• Male sex differentiation

• Secondary sex characters
• Primary sex characters
• Reproductive behavior
• Functional sex reversal
The androgenic
Hormone (AGH)

Who is behind all the above effects

in crustaceans?

Steroids?
The first androgenic
hormone AGH found

Hexahydrofarnesylacetone

Farnesylacetone
The first androgenic
hormone from isopods

B chain (44) K C peptide (46) K A chain

R R (29)

Glycan
The complete sequence of
CqIAG cDNA
ACGCGGGAGGTGCGGCACCAGCAGTGGCCCTCAGCACCCGTCCAGCACAACACCACCACT
TCATTTCTTTGCCTCTTCCCCACTGTGACTGTCCTCCTTCCCACTGTGACTGTCCTCCTT
CCCACTGCGACTGTCCTCTTCCCCATCTGTGACTGTCCTCCTCCCCCATCTGTGACTGTC
CTCCTCGCCTATCTGTGACTGTCCTCCTTCCCACTGTGACTGTCCTCCTCCTCTATCTGT
GACTGTCCTCCTACCCATCTGTGACTGTCCTCCTTCCCACTGTGACTGTCCTCCTCCTCA
5’ UTR TCTGTGACTGTCCTCCTCCCCTATCTGTGACTGTCCTCCTCCTCATCTGTGACTGGCCTC
CTCCCCTATCTGTGACTGTCCTCCTCTTCCACTCTTACCTAAAGTCACTATTCTGCTGCT
GGCTGGTTCCCCACCTCATTCAAACTGTCTTATCAGATTATTCAACTTCTAGTTTGTCTG
GACGCCAAGTAATTATCCATTACTCTCGTGGTCTCCGTCACAGCTTTACCTTCTATCAAA

Predicted
GTATAACACTCAACGATGCTGTTCCAAACATTACTCAACCTGATTTTGGTTGTGGTGGTG
M L F Q T L L N L I L V V V V
AAGCTGCCTCCTCCCTCCGCCTCTTACAGAGTGGAAAACCTTCTGATTGACTTCGACTGT
1445 bp
signal peptide K L P P P S A S Y R V E N L L I D F D C
GGCCACCTGGCGGACACAATGGACAGTATTTGCCGCACCTACCAGGAATTTAACGACACC
G H L A D T M D S I C R T Y Q E F N D T
176 aa
CGAGCGGTGAGGTCGGCCAGAGATGCATCATTTTCTGCCAGTGTCTCCATGTATGACCCC
R A V R S A R D A S F S A S V S M Y D P
GGGAGTAAGATTGCTGTTCGTCAAGTATACCATCCAAGAGGCAGGAAGTTGGGTGTCAAG
G S K I A V R Q V Y H P R G R K L G V K
TTTACTGTCCCTGATGCCAGGTTGGGTAAGCAGGAGGCGATGACAGTGAGTCGCGAGGCC
F T V P D A R L G K Q E A M T V S R E A
GCCCATACGTTTATAAAGACCCAGAACTACAACCGTCGCCGCCGTAACTCAGATACGACA
A H T F I K T Q N Y N R R R R N S D T T
GACAATACAAGCAGCACTAACGTTTATGATGAGTGTTGCAGCGAGAAAACATTGAAGACC
D N T S S T N V Y D E C C S E K T L K T
TGCGTCTTCGATGAGATTGCCCAGTACTGTGAACAGTTGGAGGACGGAATCTACGTCAGT
C V F D E I A Q Y C E Q L E D G I Y V S
TCT TGAGGTGGGTGAGCTGGAGGTAGTGACGGTGGCCTCTTTAACTCCCCTAACCATTAT
S
CAAGACACTGACCCGTCACTGCCGATGTTACATTATCAAGACACTGACCCGTTACTGCCG
ATGTTACATTGTACTCAGCAGAAACGAGCCTATTGACCACATTTATATCCTATAATTCTT
AATCAAATGTTCGTTTCTTATTATCTTATGTTGGTGTATTAATTTCTGCCTTGTTTGAAT
CTGTGACGACTTGTTTGGTTAAGCACTTTCTGTATTAGTCCAAATTTGCTCTTTTTTTAT
3’ UTR CATGACTTTCCAGCGCCTTCCATTAAATAAATTCGTATTGATCAAAAAAAAAAAAAAAAA
AAAA
The expression of CqIAG in
the male reproductive system

1000 bp
AG 500 bp

Testis CqI AG
Sperm duct (195 bp)

EFT-2
 CqIAG is a novel insulin-
like factor

B chain region A chain region

CqIAG DFDCG-HLADTMDSICRTYQEFNDTRAVRNSDTTDNTS-STNVYDECCSEKTLKTCVFDEIAQYC

Insulin of mouse QHLCGPHLVEALYLVCGE-RGFFY-TPK-----SRR-----GIVDQCC----TSICSLYQLENYC

IGF I of bovine ETLCGAELVDALQFVCGD-RGFYFNKPTGYGSSSRRAP-QTGIVDECC----FRSCDLRRLEMYC

Relaxin of human IKLCGRELVRAQIAICG----MSTWSPY------------VALFEKCC----LIGCTKRSLAKYC

Bombyxin (Silk moth) HTYCGRHLARTLADLC-----WEAGV--------------DGIVDECC----LRPCSVDVLLSYC

Insulin-like of D. melanogaster MKLCGRKLPETLSKLCV--YGFNAMTKR------------DGVFDECC----LKSCTMDEVLRYC

AGH of A. vulgare DVLCG-DIRFTVQCICNELGYFPTERLDKPCPWPNREKREIAFYQECCNIRTEHKCNRTTVSLYC

 Asn Glu Val Arg Tyr -NH2
Leu
Leu
B
Arg Asp Phe Asp Cys Gly His Leu Ala Asp Thr Met Asp Ser Ile Cys Arg Thr Tyr Gln Glu Phe Asn Asp Thr Arg Ala
Val
S S Arg

Ser
Tyr Asp Glu Cys Cys Ser Glu Lys Thr Leu Lys Thr Cys Val Phe Asp Glu Ile Ala Gln Tyr Cys
Val
Glu Ala
Asn S S
Thr
A HOOC - Ser Ser Val Tyr Ile Gly Asp Glu Leu
Gln Arg

Asp
Ser
Ser Thr Asn Asp Thr Thr Asp Ser Asn Arg Arg Arg Arg Asn Tyr Asn Gln Thr Ala
Lys
Ile Ser

Phe Phe
Thr
Ala His
Glu Ala Ser
Glu Ala Met Thr Val Ser Arg
Gly Lys Gln
Leu Ala
Arg
Ala Ser

Asp Val
Pro
Ser
Val
Met
Thr Tyr

Phe
C Asp
Pro
Lys Val Gly
Gly Leu Lys Arg Gly Arg Pro His Tyr Val Gln Gln Val Ala Ile Lys Ser
3D model of CqIAG showing the
receptor recognition surface
Gene silencing through
dsRNA

Andrew Z. Fire Craig C. Mello

2006 Nobel Prize in Physiology / Medicine

Use of RNAi in Aquaculture
(non GMO)
Mr-IAG
Insulin-like
androgenic hormone

RNA 5’- -3’

A U C U U G C A U C C U G C A U A A U G C A U A U C A C A U G A A A A A A A A A A A A A
-3’
U A C A A C C U A C G A C G U A U U A C G U A U A G U G U A C
3’- -5’

U U G C A U C C U G C A U A A U G C A U A U C A C A U G

Temporal dsRNA U A A C C U A C G A C G U A U U A C G U A U A G U G U A

A U G C A U A
effect U U A C G U A

RISC

5’- -3’
DNA A T G C A T G C T G C A T A A T G A T A T G C A T C C T G C A T A

T A C G T A C G A C G T A T T A C T A T A C G T A C G A C G T A T
3’- -5’
(Mr-IAG gene)
 Mr-IAG temporal
Silencing
Identify juvenile males:

http://www.fish.washington.edu/people/nlowry/excluder/prawnsex.html

Female
Mr-IAG silencing inhibited
appendix masculina regeneration

2nd pleopod at Start Exuvia 2nd Molt 2nd pleopod at End

Left
Silenced

1mm
Right
Left
Control
Right

Appendix interna
Appendix masculina
Ventura et al., Endocrinology 2009
Mr-IAG silencing inhibits cumulative
appendix masculina regeneration
Cumulative AM regeneration

100
Silenced
Control
80
(% / group)

60

40

20

0
0 2 4 6 8 10 12 14 16

Ventura et al., Endocrinology 2009 Time (weeks)

Mr-IAG silencing caused
spermatogenesis arrest and AG
hypertrophy

Silenced Sperm duct and AG Testis

TS

SD

50μm AG

SD
Control

sz

sz sg

AG
50μm

Ventura et al., Endocrinology 2009

 Differentiation from
fertilization to maturation
(In cultured prawns)

IAG Switch Maleness

Male
Genomic
determination (ZZ)

(WZ/ZZ)
AG formation
Female
Femaleness (WZ)

Time
Fertilization Decision point Maturation

AG- androgenic gland

IAG- Insulin-like androgenic hormone
Androgenic gland manipulation
for sex reversal
♀ ♂
WZ X ZZ

50% 50%
WZ ZZ

- Mr-IAG
Neo
ZZ

Modified from: Aflalo et al., 2006

Endocrine regulation of the
vitellogenic process in
invertebrates

The insect model

Regulation of vitellogenesis
in the mosquito

EDNH- Egg Development

Neurosecretory Hormone

MAGF-Male Accessory
Gland Factor

CCSF-Corpus Cardiacum
Stimulatory Factor
General scheme of the
endocrine regulation of
reproduction in insects
Interactions between corpora allata,
fat body and ovary in insects:
Which controls which?
Ecdysteroids and juvenoids in
plants.

Allelochemicals
 Allelochemicals
Phytoecdysteroids
Allelochemicals

Phytojuvenoids

Farnesol
Allelochemicals-Structure of
environmental estrogens

Estrogenic
Endocrine Disruptors

Xenoestrogens
Xenoestrogens -
epidemiology

Reduction in male fertility. Sperm count reduced in 50 years

from 160,000,000 / ml to 66,000,000 / ml. Current reduction rate 2% year
(England).

Prostate cancer. 67% increase between 1987-1997. Not common in

people from non industrial regions.

Breast cancer. 3 fold increase in the past 50 years (US).

From 1988-1992 an increase from 62.9 / 100,000 to 126.5 / 100,000.
6.5 fold in polluted areas.

Earlier female sexual maturity. (common

textbook information, no references)
Xenoestrogens -
epidemiology
•87,000 chemical compounds could potentially be
of concern.

•75,000 of them are in regular industrial use. Only

3% tested.

•Possible exposure :
Polluted water (industrial swage).
Dental materials.
Industrial plastic products in our surroundings.
EEDC = Estrogenic endocrine
disrupting chemicals

•All synthetic products.

•Most with an aromatic ring attached to a hydroxyl (OH) or

chloride (Cl).

•Might accumulate in
adipose tissues.

•Most have a lower affinity

to the estrogen receptor
(1:1,000,000 to 1:1,000) Estrogen receptor
Xenoestrogens:
• Nonyphenol
• DES - diethylstibestrol
• DDT- dichlorodiphenyltrichloroethane

DES

DDT
Atrizine
DDT – large scale insect control
Bisphenol A
4,4 isopropylidendiphenol
Bisphenol A (BPA) -
History
•First synthesized in the 30’ as part of a search for synthetic
estrogens.
•More potent products (DES) were found.
•Rediscovered as a cross linker for the petrochemical industry.
•Present in fungicides and general pesticides.
Present in industrial products.
 Bisphenol A (BPA)

•In Industrialized regions levels of 7-8 μg/ml could

be detected in water.
•In parts of Japan water contained 17mg/ml and
precipitation 100 μg/ml.

Half life 1 to 4 days.

•Affinity to the estrogen receptor 1:10,000

Bisphenol A (BPA)
in vivo effects

•Skin exposure causes damage to kidney, liver, spleen and

lungs (Atkinson A, 1995 (.
•Exposure causes earlier sexual maturity )K.L.
Howdshell,1999(.
• In young female rodents changes
in the ovaries and uterus
were detected
)H. Kato, 2003).

Accelerated ovary maturation with BPA 2 – 20μg/kg

Bisphenol A (BPA) prenatal
exposure (during pregnancy)

•In rodents
•BPA (2 -20 μg/kg).
•Prostate enlargement (30-36%).
•Reduction in daily sperm production.

•In human
•BPA was detected in the blood of pregnant woman.
•It seems to cross the placenta to be accumulated in the
amniotic fluid.
•BPA was found in the blood of human embryos (Ikezuki,
2002).