1/23/23

Molecular and Cellular Mechanisms of Reproduction: Sessions 1 & 2

A. Introduction to the course.

B. Normal reproductive processes

1. During a normal ovarian cycle, many follicles are recruited but
only 1 overcomes biological barriers and is selected for
ovulation.
2. The biological barriers a sperm must overcome to fertilize an egg.
3. The highly ordered process of spermatogenesis an its hormonal
regulation.

C. Infertility and Assisted Reproductive Technologies,

In vitro fertilization (IVF),
Intracytoplasmic sperm injection (ICSI)
Sperm Retrieval
and the normal biological barriers they circumvent.

Part A: Class details:

1. The Syllabus is posted in the class CoursePlus website.

See: Resources/On-line library

2. Slides and learning objectives for each lecture will be deposited

in the CourePlus website the day before each lecture.

4. All lectures will be in class. I will answer questions during or

after class. Questions can also be sent to me by email.

Classes will also be recorded via Panopto, but don’t expect

good audio.

4. Two “take home exams”. See the syllabus for details (posted in CoursePlus)
including the dates and exam formats.

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Part B.1: Normal Female Reproductive Processes

These processes impose multiple biological barriers to egg
formation and to fertilization

Selection of the ovulated follicle

Selection of sperm by the female reproductive tract
The egg’s barriers to sperm

Assisted reproductive technologies circumvent normal barriers.

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The Normal Ovulatory Cycle:

Roles of LH, FSH and estradiol in follicle
selection and ovulation.

The specific neuron in the hyp

othalamic named hypothalamic
GnRH+ Neuron will release GnRH
which is a kind of master reg Hypothalamic, Pituitary, Gonadal Axis
ulateor. This kind of neuron h 下丘脑 垂体 性腺轴
as very axis and reach to the
pituitary (垂体), and induce t 促性腺激素释放激素
he 卵泡刺激素(FSH)和黄体生成素
(LH) release into the blood.
For male, the LH will affect t
he Testis an the testis will r
elease the testosterone and bo
th repreee the activity of hap
othalamus and pituitary, but i
t does not directly affect the
hypothalamic GnRH+ neuron. Fo
r the female, when the FSH goe
s to the ovary, the ovary will
release the estradiol. Estr
adio can be active or repress
the pituiyary and hypothalamus
as well. It also could not di
rectly affect the hypothalamic
GnRH+ neuron.

The testosterone can kill some ne Only the male has LH, female has FSH or b
urones and do complex process. Th oth gender have the LH nad FSH?
e boy,therefore, usually have les
s neuron in the childhood.
6

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Normal Ovulatory Cycle: LH and FSH Regulate the Ovarian Cycle and Play Key Roles
in the Selection of the Follicle (卵泡) that Produces the Ovulat
ed Egg
LH and FSH are secreted
by the anterior pituitary.

The LH surge The high level LH will drive t

triggers ovulation he ovulation (act priamrily at
this point）

FSH stimulates follicle growth, particularly

the growth of the dominant follicle.
This occurs even though serum FSH levels
decrease during most of the first half
of the ovarian cycle.

卵巢周期
During most of the ovarian cycle, estradiol
feedback suppress LH and FSH secretion.

At midcycle, the rising serum estradiol levels

stimulate LH and FSH secretion.

Intense Selection Pressure on Oocytes

During the Reproductive Lifetime of a Woman, Her Ovarian

Cycles Kill off at Least 90% of Her Oocytes

1. At puberty, a woman has about 200,000 follicles.

2. In a young woman, 10 follicles are recruited at the

beginning of each ovarian cycle. One follicle will be
selected for ovulation. The other 9 will die by apoptosis
unless there is endocrine intervention.

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Cross Section of a Late Antral (Dominant) Human Follicle

Dominant antral
follicle selected for Is all of this folicle?
Follicle that was ovulation
recruited but not
selected for ovulation.
dies by apoptosis.

Early
Secondary
Follicle

http://medcell.med.yale.edu/histology/ovary_follicle.php

Recruitment & Selection of the Dominant Follicle

FSH-dependent LH-dependent

Atretic follicles
die by apoptosis.

10 very long progress

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The dominant follicle is stimulated by FSH.

Question:
How does stimulation occur
when serum FSH levels are
falling due to negative
feedback from estradiol?

Answer:
The dominant follicle (DM) recruits most of the ovary’s blood
supply and the FSH it contains. Other recruited follicles are
deprived of FSH stimulation.

The DM recruit about 90% of the blood supply, with the decrea
se of the FSH, the DM will recruit more and more blood
But the total of FSH given to the DM will increase?

11

Selection of the Dominant Follicle in Three Species-Measuring Follicle Sizes .

Mares
(母驴)
Sizes of the three
largest follicles
were determined
by ultrasound.

Cows

Women
: Point in time when
dominant follicle
was selected.

Ginther, O.J. et al. Biol Reprod Two different subjects

2001;65:638-647
Copyright ©2001 Society for the Study of Repr

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The Selected Follicle Recruits Most of the Ovary’s Blood Supply

Means ({+/-}SEM) for diameter (a), blood flow area (b) for the three largest follicles (F1, F2,
and F3) in mares (n = 7) with a single dominant follicle

Follicle Size

Blood flow area

Time (days)

Copyright ©2004 Society for the Study of Reproduction

Acosta, T.J. et al. Biol Reprod 2004;71:502-507

13

Follicle Selection and Vasculature

The dominant follicle develops an extensive capillary network. In contrast,
blood vessels die in follicles that are not selected (arrows)

The more blood vessicles ar

ound the DM while less bloo
d vesscles around the egg n
ot selected.
Dominant

Regressing
Cell Tissue
Res 316: 349,
2004

Bar: 200 microns 60 microns

14

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Follicle Selection and Vasculature:

The dominant follicle secretes angiogenic factors that stimulate neovascularization

Angiogenic factors:

Vascular endothelial
growth factor (VEGF)

Angiopoietin (Ang)

TIMP=Tissue inhibitor
of metalloproteinase
MMP=matrix metallo-
proteinase

VEGF stimulates endothelial proliferation and migration

Ang stabilizes of blood vessels and stimulates their sprouting.
Flt-1 and KDR: VEGF receptors Reprod. Fertil Dev. 13: 557, 2001

15 The migration and polifrication start branch off which make more epithelial cell and you have more space there. These factors not only work for the
blood vesscles formation, but also stablize the blood vesscles.

Intrafollicular FSH and Estradiol Levels in the Dominant Follicle

??????

16

People use the needle to draw the fluid form the domainant folllicle and the non-dominant follicle and measure the com
ponents in them.

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Summary: sequential negative and

positive feedback of estradiol on
the hypothalamus and pituitary.
Negative feedback: Increasing estradiol
secretion by the dominant follicle, results in
reduced FSH secretion by the pituitary, and
deprives non-dominant follicles of FSH
stimulation.

However, the blood flow to the dominant

follicle increases the amount of FSH
delivered to the follicle, FSH stimulation to
the dominant follicle increases.

Positive Feedback: At midcycle the marked

increase in serum estradiol levels stimulates LH
and FSH secretion by the pituitary. If serum
levels are sufficiently increased, ovulation
ensues.

17

Normal Cycle: Ovulation of the Egg from the Dominant Follicle:

The LH Surge Stimulates the Secretion of Proteases and Pro-Inflammatory
Cytokines by the Dominant Follicle

*
The LH Surge is triggered
by positive feedback from
increasing serum levels of
estradiol, which is secreted
by the follicle.

An insufficient LH surge
results in ovulatory failure.

* Cox-2 inhibitors, Ibuprofen and Indomethacin, block ovulation by some women.

18

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Ovulation: Changes in the structure of a rabbit follicle prior to ovulation

10 hrs prior to 30-60 min prior 1-5 min prior to
rupture of follicle to rupture of follicle
rupture of follicle

Stigma

Extravasated
RBC’s; loss
of capillaries
Disorganization
of the layer of Granulosa cells sloughed.
Capillary dilation granulosa
cells.
Surface epithelium
Thinning of tunica albuginea
mostly absent
and theca externa
Sloughing of surface epithelium
in the area of the stigma http://www.trinity.edu/lespey/m anuscripts/encyc/encyc.htm l

19 we can see the capillary and granulosa cells disorganized

The surface of the cell degraded .

Summary-Normal Ovulatory Cycle

Follicular Dominance and Ovulation

There are at least two biological barriers that a

recruited follicle must overcome for it to be ovulated :

The selected follicle must recruit most of the ovarian

blood supply.

The need for high levels of stimulation by LH.

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Biological Barriers to Natural Fertilization:

Selection of Sperm in the Female Reproductive Tract.

Uterotubal Junction
Fertilization
Occurs in the
ampulla

: Primary anatomical sites of

sperm selection

21

Selection of sperm and the

Histology of the oviduct

Crypt

Uterotubule Junction Isthmus Ampulla

The epithelium of the crypts of the isthmus This is a partial cross-section of the ampulla.
The uterotubule junction regulates
which sperm enter the oviduct. Sperm Expresses receptors for sperm. Fertilization occurs in the ampulla
The bound sperm undergo capacitation and its secretions are thought to promote
must express specific surface fertilization and early embryonic
and are gradually released into the lumen
proteins to pass this junction (e.g. development.
of the oviduct.
ADAM2, Calmegin). The uterotubule junction
Is a major biological barrier to sperm.
capaciation would do two th
The very narrow junc ings and the inr thing is c
Int. J. Dev. Biol. 52: 455,2008
tion that the sperm hange the other sperm's swi
needto cross m and the other thing is go
22 ing to allow the sperm to r
espond biochemically to spe
rm can bind to the zona pel
lucida (透明带) of human oo
cytes. The sperm bind to th
e tsthmus will cause the sp
erm go through the capaciat 11
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The Selection of Sperm: A Fertile Human Ejaculate

Contains at least 40 Million Sperm.
Very Few Enter the Oviduct/Fallopian Tube and Reach the Ovulated Egg
Reviews - Gamete and embryo transport in the Fallopian tube - H Croxatto

Table 1. Approximate number of spermatozoa at the Table 2. Number of spermatozoa recovered from
sites of insemination and fertilization. human Fallopian tubes 8–15 h post-coitum.

Species At site of At site of Range No. of women

insemination fertilization
(×106) 0 4
1–100 6
Guinea pig 80 25–50 101–1000 5
Boar 8000 1000 1001–5000 2
Man 200 10–1000
Mouse 50 20 Adapted from Croxatto (1996).
Rat 60 20–100
Sheep 1000 1000

consistent pattern of sperm migration. In women, however, genital tract that transports the luminal contents to the oviduct.
coitus may occur at any time of the cycle, or even outside of it. However, it has been established that those spermatozoa are
Therefore, depending on this timing, spermatozoa encounter not those that fertilize the oocytes (Adams, 1956). This phase
very different biological conditions in the genital tract and is followed by a slow and sustained migration that determines
diverse patterns of sperm migration are to be expected. a progressive colonization of the isthmus (Overstreet and
Nevertheless, this variability is greatly reduced, though not Cooper, 1978a, b) (Figure 2). Fertilizing spermatozoa come
abolished, if we only consider coitus occurring in the fertile from this phase (Adams, 1956).
period, i.e. which can result in pregnancy. In women, coitus
Reproductive Medicine Online 4: 160, 2002
can result in pregnancy if it occurs on the day of ovulation or It is not yet established to what extent the total number of
during any of the 5 preceding days (Wilcox et al., 1995). spermatozoa entering the isthmus is controlled by the oviduct
23 itself. It could also depend on the number of spermatozoa
Sperm migration in the female reproductive present in the previous segment that are capable of migrating.
At least in the rat, the number appears to be under hormonal
tract control (Orihuela et al., 1999) (Figure 3).

The site of insemination in some mammals is the uterus, There are barely a dozen publications reporting systematic
whereas it is the vagina in others. The latter is the case in efforts to recover spermatozoa from the Fallopian tube at
women, in whom spermatozoa migrate from semen into the known intervals following coitus in fertile women (Croxatto,
cervical mucus, where a sperm reservoir is established 1996). Failure to recover spermatozoa is not unusual, but it
(Croxatto, 1996). It is during this process that the first and does not necessarily imply there are none present. Rather, it
largest reduction in the number of spermatozoa migrating to reflects the difficulties in finding them because they are few
the next segment takes place. In animals with intrauterine and very small and have to be identified amongst the large
insemination, such reduction occurs at the UTJ. In those
Spermatozoa and the role of
species, hormones, enzymes, growth factors or cytokines
number of cells shed from the oviduct during the procedures,
all of which makes it difficult to find them. When spermatozoa
producedItsbyacrosome
the male genitalintract
fertilization.
are carried within semen, are found, the number recovered usually falls in the range of a
enter the uterus and act on uterine cells, changing their few tens to a few hundreds, reaching about one thousand in
function (Tremellen et al., 1998). only few cases (Table 2). After sexual intercourse in the fertile
period, spermatozoa continue to reach the tube from the
Apart from the aforementioned differences, several cervical reservoir for several days, but at the same time many
characteristics of sperm migration are shared among species. proceed to the peritoneal cavity and may be recovered from the
The most remarkable common feature is the enormous Douglas cul de sac. These two simultaneous processes
reduction of the number of spermatozoa reaching the site of probably determine that the overall number of spermatozoa
fertilization as compared with the number ejaculated (Harper, inside the tube remains low and constant during the days
1994) (Table 1). In spite of that reduction, the number is still preceding ovulation. There is yet no evidence for the
too high if one considers the number of oocytes available for establishment of a reservoir of spermatozoa in the isthmus in
fertilization. Given the fact that the population of non- the human (Williams et al., 1993), comparable to that observed
fertilizing spermatozoa is huge and outnumbers by far the in several other species (Hunter, 1988).
minimum number required to achieve fertilization, it has been
proposed that such a population could serve other functions, Details of the spatial distribution of spermatozoa within the The sperm is smal
such as inducing optimal conditions in the genital tract to
render the reproductive process more efficient. Some
tube are poorly understood. A brief examination of the lumen l and easy to swi
of the human tube shows that the virtual space in which they m
experimental and clinical observations support this hypothesis can distribute is an immense and intricate maze drawn by the
(Geva et al., 2000; Tremellen et al., 2000). numerous and complex folds of the mucosa that invade the
lumen, specially in the ampulla (Figure 4). Even the most
Another typical feature is that spermatozoa reach the oviduct optimistic estimate suggests that chances for male and female
just a few minutes after coitus, covering a long distance in a gametes to meet inside the Fallopian tube must be very low in
.
very short time, which would not be possible if it were due the absence of ad hoc mechanisms that facilitate their
only to their own motility. This is the so-called rapid phase of encounter. Such mechanisms could be the large contact surface
162 sperm migration and it is probably due to contractions of the offered by the expanded cumulus oophorus, a persistent

24

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Part B2.
Biological Barriers to Fertilization:
The zona pellucida
Sperm must bind and penetrate the zona pellucida, fuse
with the egg plasma membrane and activate embryo development .

Zona the sperm can use the enzyme and dig to ho

Pelucida le and go into the egg through the acrosom
The init e reaction .
ial site
binding
the spe
rm to th
e egg

25

when the sperm bins to the A Biological Barrier to Sperm at fertilization: The Acrosome Reaction
zona pelucida, it will ca The acrosome reaction occurs when sperm bind to the zona pellucida
use the signaling transdru Only an Acrosome-reacted sperm can fuse with an egg’s plasma membrane
ction in the sperm which c
ould cause the fusion of t
he plasma membrane and the
outer acrosomal membranes.
Then, the acrosomal cont
ents will release and degr
ade the eggs protection la
yer.

26

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A Biological Barriers to Fertilization by a second sperm: Egg activation

egg activation Increased cytoplasmic Ca+2 levels
which can make stimulate the egg to release its
sure only one s cortical granules. These degrade
perm can frtili The ZP’s sperm receptors. No
other sperm can bind the ZP.
ze with the egg,

EGG

Egg activation is initiated by

Phospholipase C zeta (PLCz)
from the sperm. This enzyme
hydrolyzes PIP2 to IP3

IP3 triggers oscillations in the

Zona Pellucida concentration of free Ca+2 in the egg’s
cytoplasm. This depolarizes the egg plasma
membrane, blocking the ability of the egg
to bind additional sperm.
Reproduction 152: R41, 2016

27

the start of embryo genesis.

The final barrier to fertilization: The Sperm must induce calcium oscillations
in the egg.
These oscillations are required for the fertilized egg to initiate embryogenesis

The increase of the Ca2+ is the signal of the embryo progress begin.

IP3 regulates calcium release from an egg’s ER. The probability

that an egg will activate is determined in part by the numbers of
calcium oscillations that occur in response to fertilization.

* Measure of Fluorescence These oscillations do not always occur when fertilization is

achieved by intracytoplasmic sperm injection. Then, Ca
oscillations then must be induced by depolarizing the plasma
Reproduction 152: R41, 2016 membrane

28

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Summary-Oogenesis, Ovulation and Fertilization

1.Ten early antral follicles are recruited at the beginning of each ovarian cycle of a
a young, fertile woman. One follicle will become dominant and ovulate.
FSH and LH are essential regulators of these processes.

2.The dominant follicle recruits most of the blood supply to the ovary. This insures
increasing FSH stimulation even though serum FSH levels are decreasing.

3. A robust mid-cycle increase in serum LH levels triggers ovulation.

4. The female reproductive tract imposes a number of biological barriers to sperm.

Consequently only 10-1000 sperm reach the ovulated egg.

5. The egg also imposes biological barriers: sperm must bind and penetrate the
zona pellucida, undergo the acrosome reaction, bind to and fuse with the
egg plasma membrane and the activate the egg.

29

Part B3: Spermatogenesis and Its Hormonal Regulation.

30

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Spermatozoa are highly

differentiated, morphologically
unique cells that are produced
by precise molecular and
cell biological processes.

spermatozoa
the maturemotilemale sex cell of an
animal, by which theovumisfertilized,
typically having a compact head an
d one or more longflagellafor swimm
ing

31

Spermatozoa are highly

differentiated, morphologically
unique cells that are produced
by precise molecular and
cell biological processes.

A young fertile man produces

between 500-1,000 sperm per
second.

32

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Spermatozoa are highly

differentiated, morphologically
unique cells that are produced
by precise molecular and
cell biological processes.

A young fertile man produces

between 500-1000 sperm per
second.

This high level of gamete

production insures that
a fertile ejaculate contains >
40 million sperm.
A man with a sperm
count lower than this is usually
. infertile.
Large and continuous production of the sperm.
33

The Process of Spermatogenesis: Large Scale Gamete Production (Illustrated for the Mouse)
Meiosis
Homologous Recombination / Double Strand Break repair The testis
expresses
higher levels of
DNA repair
enzymes
Mitotic proliferation of spermatogonia

than any other

organ.

Base Excision
Repair
Spermiogenesis

Sequential
Replacement of
Histones with
With Protamines

Sperm atogonial stem cells

and transit am plifying
progenitor sperm atogonia

34 Differnt process and number among species

Duplicate the chromation rather than the chromosome
During the meiosis , the recombination is required otherwise the cell will die
Before the morphology changes and the repire of DNA will happen again, if you expose the tonxic things for a long tine, it
would be destory the repire system.
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Proper Compaction of Spermatid Nuclei Is Required for Formation of Fertile Spermatozoa

Spermatid nuclei before compaction Spermatid nuclei after compaction

Nucleus
Nucleus

Nucleus

Sperm DNA is so tightly packed that it is not transparent to an electron beam. David Phillips

35 The nucleus will very compated. The histone in the dna removed
A pazzle: id the removal of the histone will cause the physical break of DNA?

The structure of chromatin in spermatogonia, spermatocytes and

round spermatids as well as in somatic cells

highly campated chrotain;gene slience; the differentation of the cell, important

Nucleosomes
are tightly packed.
The epigenomes of
DNA not accessible heterochromatin and
to transcriptional euchromatin differ.
machinery the methalated chromation are not charge

Heterochromatin:
DNA - CpG dinucleotides two differences between heterochroma
methylated tin and euchromatin
. Histones – methylated
C-terminal tails.

Euchromatin:
Nucleosomes
dispersed. DNA DNA- not methylated
accessible to Histones – acetylated C-
transcriptional
Nucleosome: 2 copies each machinery terminal tails.
of Histones H2A, H2B, H3
and H4

The access of the gene which can be transcriped

36

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Nuclear Compaction of Sperm Requires Marked Changes in Chromatin Structure

Replacement of Histones with Protamine

During Spermiogenesis

how t
o ill
ustra
te th
is fi
gure?

Chromatin in Sperm:
Protamine-DNA Toroids plus a small
amount of DNA packaged in nucleosomes
Toroid formation condenses the nucleus

Kidney Research and Clinical Practice 31: 139, 2012

The Sperm Cell, Second Edition, Cambridge University Press, 2017
Molecular Human Reproduction 2: 929, 1996

The gold particles can remove the histones that could

37 Gold nano-particles firstly can reduce the sperm parameters such as
motility and normal morphology and secondly affect sperm chromatin make the DNA condnsed.
remodeling and cause the increase instability of chromatin and also in
crease the rate of sperm DNA damage. These deleterious effects wer
The area of the genome association wit
e more obvious in maximum dose and chronic phase. h the histone and protamine and the pa
cking is no random. the chromatin stru
cture in the fertile and unfertile spe
rm are quite different
Not All Sperm DNA is Packaged in Protamine. Some Remains Bound to Nucleosomes
Infertile sperm often have aberrant DNA packaging.

The DNA and histones in these solenoids form nucleosomes.

Questions:
Are specific genes packaged in these nucleosomes?
Do the epigenetic marks on the DNA and histones in these
nucleosomes suggest anything about the role of these genes
in early embryogenesis?
What are the consequences of incorrect packaging of sperm DNA or the
presence of abnormal epigenetic marks on sperm DNA or histones?

38

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Improper Packaging of Human Sperm Nuclei by Protamine

Increases Numbers of Double-Strand DNA Breaks

DNA Fragmentation
Index

J. Andrology 26: 741, 2005

39 The lower of the protamine concentration will result in the higher po

sibility of the DNA breaks. The important of the replacement of the p
roramine.
What regulates Sperm
atogenesis? In the most
mammals, the males
only breed when the f Spermatogenesis is Dependent on Androgens and on Follicle Stimulating Hormone.
Sertoli Cells Mediate the Effects of These 2 Hormones on Spermatogenesis.
emales are ???.
The testosterone is
the immportant in the The sperm form (where) i
sex behavior especia t form next to a very olyze
lly the agressive beha red cell called the sertoli
viors. The male usual cell
y fight with each other.
and lose body weight.
One of the thing happ
en to the biology is th
at the testosterone The sertoli cell synthesis
is important for the sp the basement membra
erm genesis. ne

The basement memebra

ne have a lot of informati
on to direct the cell beha
viors such as whether th
e cell devide or not.

40

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Sertoli cells are the primary mediators of the effects

of androgens on spermatogenesis.
Effects of Sertoli Cell-Specific and Global Knockouts of the Androgen Receptor Gene

Testis Histology The cell is much great

er and highly brached
Wild Type Sertoli Cell-Specific KO Global KO

Sertoli cell-specific KO via Cre-Lox.

Global KO: AR excised in embryonic stem cells which
Chang C et al. PNAS 2004;101:6876-6881
are then injected into mouse blastocysts.

41

Effect of Sertoli cell-

specific AR knockout
on germ cell proliferation
and death.

Chang C et al. PNAS 2004;101:6876-6881

42

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Some Men Without A Functional FSH Receptor Are Fertile

43

How Does A Mammalian Testis Produce Such Large Numbers of Sperm?

Spermatogenic Cells Are Organized Spatially by the Somatic Sertoli Cell
in the Seminiferous Epithelium

Blood-testis Barrier
Isolates developing
germ cells from the
Immune system.

44

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Spermatogenic Cells are Organized Temporally

In the Stages of the Cycle of the Seminiferous Epithelium
4 of the 14 Stages of the Rat Cycle: Each Sertoli cell is surrounded by
4 generations of developing spermatogenic cells

Time

NOTE CHANGES IN POSITIONS OF SPERMATOGENIC CELLS AS STAGES PROGRESS

45

Stages of the Cycle of the

Seminiferous Epithelium of the Rat
The germ cells surrounding a Sertoli cell progress from one stage of
the cycle to the next. The next stage after stage XIV is stage I.

These spermatids are released into the lumen of

the seminiferous tubule at the end of stage VIII.
Spermatogenesis

Stages I to XIV
Time

spermatogonia spermatocytes spermatids

46

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Stages of the Cycle of the

Seminiferous Epithelium of the Rat
The germ cells surrounding a Sertoli cell progress from one stage of
the cycle to the next. The next stage after stage XIV is stage I. Thus,
the stages are cyclic.

These spermatids are released into the lumen of

the seminiferous tubule at the end of stage VIII.

Stage:I to XIV

47

Spermatogenic cells and Sertoli Cells Interact Extensively

48

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PNAS 105: 8315-8320, 2008. CELL-SPECIFIC AND STAGE-

SPECIFIC GENE EXPRESSION BY
RAT SPERMATOGENIC
AND SERTOLI CELLS

98 Probe Sets

318 Probe Sets

677 Probe Sets

Stage-specific gene
expression by Sertoli cells-
regulated by signals from
developing spermatogenic 193 Probe Sets

cells.

49

Stage-Specific Changes in Replication and Differentiation Spermatogonial Stem

Cells (SSCs) Are Regulated by Cyclic GDNF Expression by Sertoli Cells.

If Sertoli cells express constant

high levels of GDNF, SSCs do not
differentiate; they form
a benign germ cell tumor.

Biol. Reprod
85: 763, 2011

Reproduction
121:347,2001

50

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Reasons that spermatogenesis can be so efficient

The process of spermatogenesis is organized both spatially

and temporarily. Immature sperm are being constantly released.
Spermatogenic cells interact extensively with Sertoli cells.
As the stages of the cycle progress, gene expression by Sertoli cells
changes.
Some of the products of these genes are essential for normal
spermatogenesis.

Spermatogenic
cells

Sertoli Cells

These interactions occur at a cellular,

Physiological, biochemical and molecular level.

51

Summary –spermatogenesis.
1 Spermatogenesis is a highly efficient process that produces large numbers of
a highly differentiated, motile gamete.
2. Most sperm DNA is compacted by protamine into toroids. However,
some DNA remains associated with nucleosomes. Aberrant compaction is
associated with DNA strand break and infertility.
3. Spermatogenesis is efficient because of the spatial and
temporal organization of spermatogenic cells within seminiferous
epithelium.
4. Sertoli cells organize the spermatogenic cells within the seminiferous
epithelium.
5. Sertoli cells interact extensively with spermatogenic cells. A consequence
of these interactions is stage-specific changes in gene expression
by Sertoli cells. Proper expression of some of those genes
is essential for male fertility.
6. Inadequate sperm production is one cause of male infertility

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Part C. Fertility, Infertility and Assisted Reproductive Technologies

United States Data (2016)

3,875,110 live births from natural conceptions

65,996 live births conceived by infertile couples by use of an

assisted reproductive technology (1.7% of all births)

Definition of infertility: failure of a couple to conceive after one year of

unprotected sex.

12-15% of couples meet this definition.

Infertility by sex:

9% of men
11% of women

CDC
https://www.nichd.nih.gov/health/topics/infertility/conditioninfo/common

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Causes of male Infertility:

Blockage of ducts from testis.

Inadequate hormonal stimulation
Inadequate sperm production

Definition of an infertile ejaculate:

< 15 million sperm / ml of ejaculate
< 39 million sperm / ejaculate*

* Mayoclinic.org

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Why Are So Many Sperm Needed for Fertility?

VS.

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Assisted Reproductive Technology (ART)

Intrauterine insemination.

GIFT

In vitro fertilization

Intracytoplasmic sperm injection (ICSI)

Micro-testicular sperm extraction.

How do IVF and ICSI differ from natural fertilization?

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Natural Cycles vs ART Cycles

The Natural Cycle

Peak serum LH concentrations

occur within 17 hours of the
initiation of the surge.

Ovulation occurs 10-12 hours

thereafter.

In almost all cases, 1 oocyte

is ovulated per cycle.

Comput Biol Med 25: 405, 1995

Reprod Med 28: 402, 1983.

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Standard ART Cycle

ART cycle: Hormonal manipulation of the ovary and
Circumvention of the normal recruitment/selection process

From: Chapter 20. Treatment of the Infertile Couple

Williams Gynecology, 2e, 2012
Human Reproduction 33: 58, 2018 J. Clin Endocrinol Metab 86: 2607, 2001

hCG or rLH

GnRH agonist or antagonist

rFSH

3 to 30 eggs retrieved by aspiration of follicles

Aspiration occurs 32 to 38 hours after hCG- or rLH- stimulation

Date of download: 3/20/2014 Copyright © 2012 McGraw-Hill Medical. All rights reserved.

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Natural reproduction vs. IVF.

Natural Fertilization Conditions
1 egg
Insemination and ovulation
coordinated
10-1000 sperm reach the ampulla
of the oviduct, the site of
fertilization.

IVF conditions
1-3 eggs
72,000 to 90,000 sperm

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Requirements for success.

Natural Fertilization
Insemination and ovulation coordinated.

The recruitment, selected and ovulation

of an oocyte are regulated by a the normal
female endocrine cycle.

In a normal cycle, one oocyte is ovulated; 9

fail the selection process and die.

Sufficient numbers (> 40 million) motile,

fertile sperm in ejaculate.

Fertilization leads to normal embryo

development

IVF
A woman’s endocrine cycle is blocked
pharmacologically. Oocyte recruitment and
selection is induced by hormone
administration.

Multiple metaphase oocytes are retrieved

from woman’s ovary.

Some motile sperm in ejaculate.

Sperm are able to fertilize and activate an
egg.

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Intracytoplasmic sperm injection (ICSI): The second major

advance in Assisted Reproductive Technology.

Requirements for successful ICSI:

Sperm recovered from ejaculate, epididymis, vas deferen or testis.
Sperm need not be motile or morphologically normal.
Metaphase I oocyte recovered from woman.
Egg is activated by sperm or by electrical depolarization of egg.
Embryo develops.

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Major differences between natural fertilization and IVF or ICSI

1.In both IVF and ICSI the woman’s natural cycle is blocked and
development of multiple follicles (3-30) driven by injection of FSH
and LH. Thus, both IVF and ICSI circumvent the normal follicle
selection process.
2.Both IVF and ICSI eliminate the biological barriers that the female
reproductive tract presents to sperm.
3.The ratio of sperm to eggs is logs higher in IVF.
4.ICSI eliminates the biological barriers presented by of the zona
pellucida and the egg plasma membrane.
f.The biological barrier of egg activation by sperm may be circumvented
during ICSI.

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IVF in the United Stated (2012): Numbers of Cycles and Success Rates

All causes of a woman’s infertility

Woman’s Age < 35 35-37 38-40 41-42 >42
# Cycles 54,329 27,483 24,067 12,462 7,756
% Live Births 41% 34% 25% 15% 6.6%

Infertility of the Man

Woman’s Age < 35 35-37 38-40 41-42 >42
# Cycles 12,876 5,359 3,268 998 368
% Live Births 46% 42% 28% 17% 13%

In 2012 in the United States

148,956 IVF cycles
~ 50,000 children conceived by ART in the U.S.
~20% of ART cycles are because of male infertility.

Source: Society for Assisted Reproductive Technology

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Sperm retrieval from an infertile man

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Sperm may be aspirated

from the epididymis or the
testis of an infertile man.
These sperm may be used
to fertilize an egg by IVF, if
sperm were collected from
the cauda epididymis, or by
ICSI.

Sperm mature and gain

motility and fertilizing
capacity as they move
through the epididymis.

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A last resort: Micro-Testicular Sperm Extraction (TESE):

Surgical
An isolated extraction
region of a seminiferous
of normal-appearing tubule with
tubules with spermatogenesis sperm.
outlined by arrows.

Requirements for successful micro-TESE

Sperm are present in at least one segment of one testis tubule.
Segment of tubule can be accessed surgically, removed by dissection
Peter N. Schlegel
Hum. Reprod.
and sperm retrieved.
1999;14:131-135 Fertilization by ICSI.

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IVF AND ICSI circumvent barriers inherent in the

development of the dominant follicle, and the
barriers normally imposed on sperm by the
female reproductive tract and the egg.

TESE circumvents the processes of sperm

maturation.

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