Raul Martínez-Zaguilán

Testicular Function

.
.

Neuroendocrine Control of Testicular Function

I. Structure of the testis

Seminiferous Tubules
Sertoli Cells
Spermatogenic Cells
Interstitial Compartment
Leydig Cells

Rete Testis

Epididymis

Accessory Glands (seminal vesicles, prostate)

II. Functions of the testis

Spermatogenesis
Primary Spermatocyte
Secondary Spermatocyte
Spermatid
Spermiogenesis

Steroidogenesis

III. Somatic Cells

Leydig cells

Sertoli Cells

IV. Steroid Hormones in Circulation

Testosterone
V. Neuroendocrine Regulation of Testicular Function.
Regulation of Release.

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Testicular Function

I. Structure of the testis.

The two major functions of the testis are production of the male germ cell (sperm), or
spermatogenesis, and production of male sex steroids, or steroidogenesis. These two functions are
compartmentalized within the testis. The testes in man are paired organs located in the scrotum,
outside the abdominal cavity. This location of the testes is essential for spermatogenesis because
this process is temperature-sensitive and cannot occur at normal body temperature. The
temperature differential between testes and abdominal cavity is maintained, in part, by the close
apposition of the spermatic artery and vein so the incoming arterial blood is cooled by the colder
venous outflow. Each testis is divided into a number of pyramidal septa which are separated by a
connective tissue capsule. Each septum contains a large number of tubules, the seminiferous
tubules, which are convoluted so that they occupy a minimal amount of space within the septum
(Fig 1). The seminiferous tubules are avascular and are the site of spermatogenesis and sperm
transport. They are composed of a tubular wall formed by peritubular cells resting on a basement
membrane that encloses both Sertoli cells and spermatogenic cells. The latter referred as germinal
epithelium because sperm cells are produced there (Fig 2). The seminiferous tubules empty their
contents into the rete testis through which the
contents move to the epididymis fro providing
some sperm maturation and sperm transport
along the reproductive tract (cf., Fig 1). It is
worthwhile to mention however, that final
sperm maturation and the ability to fertilize an
egg occurs in the female reproductive tract, a
process called sperm capacitation.
Interestingly, sperm can remain viable in the
female reproductive tract for ca. 48 hrs, thus
increasing the probability of fertilization. The
interstitial compartment contains the Leydig
cells, lymphatics, blood vessels, and various
connective tissue elements and occupies the
space between seminiferous tubules. The
interstitium is the site of steroidogenesis (in
Leydig cells) and is highly vascularized.
Therefore, testicular compartmentation affords
the ability of mature sperm to be selectively
transported outside of the body, and the steroid
hormones to have localized (i.e., paracrine)
effects on spermatogenesis, but also to
communicate with distant sites within the body
via circulation (i.e, endocrine regulation).

Approximately 80% of testicular mass consists

of the seminiferous tubules (cf., Fig 1).
Examination of these tubules in cross section
Figure 1 Schematic representation of the human testis.

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Testicular Function

(Fig 1) reveals anatomical progression that

parallels the temporal sequence of
spermatogenesis. Sertoli cells lining the
basement membrane of the tubules provide
crucial support for spermatogenesis. Also
lining the basement membrane are
undifferentiated Spermatogonia with each
clone of differentiating daughter cells
migrating toward the tubule lumen as it
moves progressively through the stages of
spermatogenesis. A detailed schematic
representation is shown in Fig 3.

A major function of Sertoli cells is to

maintain tight junctions between themselves
and thereby form the blood-testis barrier.
This barrier separates the basal and
adluminal compartments within the
seminiferous tubules, and provides avascular Blood Vessel
compartmentation for the tubular lumen.
The barrier prevents the movement of
protein, charged organic molecules, and ions
from interstitial fluid into the seminiferous
tubules. Consequently, the composition of
intratubular fluid differs markedly from
interstitial fluid, and this composition may Figure 2 Histological section of human testis.
be critical for later stages of
spermatogenesis. The blood-testis
barrier also provides important
immunological protection,
preventing the formation of
antibodies against the highly
differentiated spermatozoa.

II. Functions of the Testis.

Spermatogenesis.
Spermatogenesis involves the
complex process by which
relatively undifferentiated germ
cells containing a diploid
Figure 3 Diagrammatic representation of the Sertoli cell and
component of 46 chromosomes
progression of spermatogenesis in the seminiferous tubule.
are turned into extremely

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Testicular Function

specialized sperm in which the

chromosome number is reduced
by half (haploid) through
meiotic division. This halving of
the diploid chromosome number
occurs prior to the complete
differentiation of spermatozoa
(Fig 4). Meiosis also occurs in
the ovum. Therefore,
subsequent pairing of male and
female chromosomes at the time
of fertilization has the effect of
giving each individual unique
genotype. Spermatogenesis (i.e,
formation of spermatozoa from
a spermatogonia) in humans
requires 64 days; however the
time between initiation of two
successive spermatogenic
generation is ca. 16 days. Thus, Figure 4. Spermatogenesis. A primary spermatogonia undergoes mitosis
when one clone of daughter cells to produce primary and then secondary spermatocytes in preparation for
is a quarter of the way through meiosis. This last process results in spermatids and spermatozoa
gametogenesis, the next clone containing a haploid set of chromosomes.
begins to form, and four
successive clones, each further along
in development and closer to the
tubular lumen. Are visible in a single
histological specimen. The rate of
spermatogenesis is hormone
independent and consistent within a
species. However, the yield is
hormone-dependent and varies from
one individual to another.

Spermatogenesis can be divided into

three major stages: (a) mitotic
proliferation, (b) meiosis, and (c )
packaging (or spermiogenesis; cf., Fig
3). It begins with mitosis o of a
spermatogonium to form two
daughter cells. One of these cells then
continues to go on to form mature
sperm, while the other is retained at
Figure 5 Meiosis allows the reduction in the number of
chromosomes to half and exchange of chromosome segments in
preparation
4 for fertilization.
 Raul Martínez-Zaguilán
Testicular Function

the basement membrane as a ready source for maintaining the germ cell line. After undergoing a
number of mitotic divisions, the primary cells becomes a primary spermatocyte and enter a resting
phase during which chromosomes are duplicated in preparation for meiosis. In man, the daughter
cell divides once to become a secondary spermatocyte, and again to become a spermatid. These
two meiotic divisions are without DNA replication, and therefore, four haploid (23 chromosomes)
spermatids are produced from one primary spermatocyte (cf., Fig 4; Fig 5). In addition to the
reduction in chromosome number, meiosis allows to exchange segments of DNA, thus mixing
their genetic information. Even after meiosis, spermatids resemble normal epithelial cells.
Production of mature spermatozoa requires extensive remodeling of both nucleus and cytoplasm.
This process is known as spermiogenesis or packaging and involves: (a) condensation of
chromatin into a tight inert packet, (b) formation of acrosome by aggregation of Golgi-produced
enzymatic vesicles, (c ) growth of the tail out of a centriole, and (d) extrusion of most of the
cytoplasm.

III. Somatic Cells.

There are two important somatic cell types in the testes which provide a crucial function to male
reproduction: Leydig and Sertoli cells.

Leydig Cells. These cells are located in the vascularized interstitial compartment and are the site
of synthesis and secretion of the male sex steroid
hormones testosterone and androstenedione. These
hormones are known as androgens. These cells
respond to luteinizing hormone (LH), which is TARGET CELL
produced by the anterior pituitary, producing Nucleus
androgens. LH is required form maintenance of
testicular tissue. In males deprived of adequate LH
levels (such as those on anabolic steroids which
provide negative feedback to the pituitary),
reversible testicular atrophy can occur. In addition Proteins
to testosterone, the testes secrete androstenedione,
T
and dehydroepiandrosterone (DHEA), bu because DHT
of their relatively low secretory rates and Cell Growth
T
potencies, in terms of testicular function,
testosterone is the major steroid that needs to be
considered. Moreover, the adrenal cortex is the
major source of DHEA and androstenedione in
Figure 6 Biochemical action of testosterone.
both men and women. Once secreted, testosterone
Testosterone crosses the plasma membrane and is
is released into the interstitial fluid where it can
converted to DHT, which then binds first to a
freely diffuse across the basement membrane and
cytosolic receptor. The hormone-receptor complex
into the seminiferous tubules to produce local
bind then to the DNA where it induces mRNA
effects on sperm production. Testosterone can also
encoding for specific proteins.
diffuse into nearby lymphatics and blood vessels to

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Testicular Function

produce systemic effects.

Sertoli Cells. These cells are located within the seminiferous tubules and perform several tasks
essential for normal spermatogenesis. The exhibit receptors for the other pituitary gonadotropin,
follicle stimulating hormone (FSH), which serve to regulate Sertoli cell function. Sertoli cells are
located in an avascular compartment; however, since they provide formation of the blood-testis
barrier, the basal face communicates with the vascularized interstitium.

The major function of the Sertoli cells are:

1. Maintain the blood-testis barrier
2. Clean-up damage cells by phagocytosis
3. Provide nourishment for developing sperm
4. Maintain appropriate ionic composition of the seminal fluid
5. Serve as a target for hormonal regulation of spermatogenesis
6. Produce estrogens from androgens
7. Synthesize and secrete specific proteins: androgen binding protein (ABP) and inhibin

ABP is important for spermatogenesis because it acts as a transport protein for testosterone both
in the Sertoli cell and following its release into the lumen of the reproductive tract. High
concentrations of ABP and testosterone are found in the epididymis, where the action of
testosterone is required for maturation of sperm. Thus, ABP helps to sequester testosterone
within the tubular lumen and prevent its free diffusion back out of the seminiferous tubule.

Inhibin is a protein that feeds back to selectively inhibit FSH, and not LH secretion from the
anterior pituitary. This provides a specific form of regulation that can preferentially alter secretion
of the gonadotropin on an individual basis.

IV. Steroid Hormones in Acetate

Circulation. Cholesterol
CH3 CH3
C O
The major male sex steroid hormone C O
OH
O

produced by the testes is testosterone.

testosterone, like other steroid hormones, is OH
OH
lipophilic, and therefore once release into OH
Pregnenolone
17-a-Hydroxypregnenolone Dehydroepiandrosterone
CH3 OH
the blood circulates largely (97%) bound to CH3
C O C O
serum binding proteins. Up to 47% is OH
bound to sex steroid binding globulin OH
(SBG), also known as testosterone binding Androstenediol
O O
17-a-Hydroxyprogesterone
globulin (TeBG), which is produced in the O
OH
Progesterone
liver; whereas 50 % is bound to albumin.
Therefore, only 3% of testosterone is free.
O
Only the free testosterone is the active form O
Testosterone
Androstenedione
of the hormone. Once secreted,
Figure 7 Biosynthesis of androgens.
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 Raul Martínez-Zaguilán
Testicular Function

testosterone can arrive at distant sites and be further metabolized. Testosterone can be converted
to two active metabolites [i.e., dihydrotestosterone (DHT) and estradiol], as well as an inactive
metabolite (androsterone 17 keto-steroid (Fig 7, Fig 8). In sex accessory tissues, testosterone
diffuses into the cell and is converted to DHT by the enzyme 5 reductase (Fig 8). This is a
membrane-bound enzyme found primarily in the male reproductive tract and liver. At these sites,
DHT is more potent than testosterone in stimulating reproductive tract growth. While this
conversion is a critical step in the accessory sex glands, it may not be as important in many of the
other actions of androgens. Conversion of testosterone by the enzyme aromatase to another active
metabolite, estradiol, occurs in peripheral tissues (cf., Fig 8). Therefore, testosterone can be
viewed as a pro-hormone because, it can also serve as a precursor for two other active steroid
hormones (i.e., DHT and estradiol).

Physiological Effects.

The major physiological effect of testosterone relates to its absolute requirement for
spermatogenesis. In addition to this, it produces a variety of reproductive and non-reproductive
effects:
1. Promotes differentiation of the reproductive tract in the developing male fetus (vide supra).
2. Provides maturation of the reproductive tract at puberty and maintenance in adults of the
reproductive tract
3. Responsible for development of secondary sex characteristics in the male, at puberty, including:
a) male pattern of hair growth
b) lowering of the voice
Dihydrotestosterone
c) anabolic effects on muscle mass
d) Growth spurt and epiphyseal closure at OH

puberty
e) increase secretion of sebaceous glands
associated with acne O H
4. Development of sex drive at puberty OH
5. Regulation of pituitary release of gonadotropins
by negative feed back OH
O
The physiological significance of androstenedione
is not fully understood. However, it may be Testosterone
OH
required for growth of the secondary sex
Estradiol
structures. DHT, in contrast, is known to be more
effective than testosterone in producing fetal Figure 8 Metabolism of testosterone.
development of the prostate, penis and scrotum,
and in promoting sperm maturation and transport in the adult. High levels of DHT are synthesized
in several tissues, including:(a) skin, (b) prostate, (c ) seminal vesicles, and (d) liver.

Testosterone is converted to estrogens in Sertoli cells an peripheral tissues including: (a) brain, (b)
skin, (c ) adipose tissue, and (d) liver (cf., Fig 8). Estrogens levels in males are in similar range

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with those in females (during certain times of the menstrual cycle; Table 1).

Serum Steroid Conc. (ng/dl) Male Female

Testosterone 700 40
Androstenedione 120 170
Dihydrotestosterone (DHT) 45 20
17 -estradiol 0.60 0.6-7.0

It is apparent from table 1 that these levels are similar in both sexes with the exception of
testosterone, which is more abundant in males than females. Thus, it is believe that rather than
absolute levels, it is the high levels of testosterone to estradiol ratio in males that prevent
feminization. The physiological significance of estrogens in males is not clear, but it can provide
selective feedback regulation on gonadotropin release from the anterior pituitary.

V. Neuroendocrine Regulation of
Testicular Function.
Regulation of Release.
The hypothalamic hormone FSH-LH
releasing hormone (also known as
gonadotropin releasing hormone, GnRH)
stimulate secretion of LH and FSH from
gonadotropes in the anterior pituitary. The
primary target of LH is the Leydig cells in
the interstitial compartment, that secretes
testosterone in response to stimulation (Fig
9). The primary testicular target for FSH
is the sertoli cells, located in the
seminiferous tubules. Upon stimulation,
Sertoli cells provide support for developing
germ cells during spermatogenesis (vide
infra). Testosterone provides negative
feedback on LH and FSH release at the
level of the hypothalamus and pituitary (Fig
10). In contrast, inhibin produced by
Sertoli cells, provides selective inhibition of Figure 9 Biochemical actions of LH and FSH on Leydig
FSH release from the pituitary. Critical to cells and Sertoli cells, respectively.
maintenance of testicular function is the
ultradian pattern (1 pulse/70-90 min) of GnRH release from the hypothalamus, such that exposure

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Testicular Function

of the pituitary to continuously high levels of GnRH

inhibits rather than stimulates gonadotropin
release. The pulsatile nature of GnRH secretion has
been inferred from measurements of LH
concentrations in peripheral blood in humans (Fig
11). It is worthwhile to mention that the
concentrations of FSH tend to fluctuate much less
because FSH has longer half-lifes than LH. In this
regard, agonist of GnRH continuously administered
can be as effective as antagonist at reducing
testicular activity. The gonadotropins also are
released in a circadian pattern with a small peak in
the early morning. This is not of particular
significance in adults; however, at the time of
puberty, nocturnal pulses of increased LH and
testosterone secretion can be measured as an
indicator of increasing testicular activity (Fig 12). Figure 10 Neuroendocrine regulation of testicular
function. Since the effects of testosterone on
adenohypophysis are unclear, they are indicated by a
dashed line.

Reading Required:

1. Goodman, M. Basic Medical Endocrinology. 2nd Ed., Lippincott-Raven Publishers, 1994., p.

175-202.

Additional Reading:

1. Guyton, A.C. Textbook of Medical

Physiology. 8th Ed., W.B. Saunders
Co., 1991., p. 885-897.

2. Berne R.M., and Levy, M.N. Physiology.

3rd Ed. C.V. Mosby Co., 1993, p.980-1000.

3. Ganong W.F. Review of Medical

Physiology. 16th Ed. Lange Medical
Figure 11 LH secretory pattern monitored in a 36
Publications. 1993. p 387-395
year old man.

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 Raul Martínez-Zaguilán
Testicular Function

4. Hadley M.E. Endocrinology. 2nd Ed. Prentice Hall. p 409-427, 1988.

Sleep
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16
14
12
10
8
6
2D Graph 1
4
350
300
250
200
150
100
50
0
2200 0200 0600 1000 1400 1800 2200
Clock Time

Figure 12 Nocturnal pulsatile secretion of GnRH is indicated by the

plasma levels of LH and testosterone in a pubertal 14 year old boy.

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 Raul Martínez-Zaguilán, Ph.D.

Sex Determination and Sexual Differentiation.

Raul Martinez-Zaguilan, Ph.D.

1. Sex Determination

2. Differentiation of the Gonads

3. Differentiation of the Internal and External Genitalia

4. Differentiation of the Nervous System

1. Sex Determination. The sexual

genotype of an individual is determined at
the moment of the conception. This is
determined by two of the 46 chromosomes
present in all human cells. If these two sex
chromosomes are XX, the individual is
genetically female; if they are XY, the
individual is male. Since the haploid (23
chromosomes) germ cells of females
contain only X chromosomes, the ovum
must also contain an X chromosome (Fig
1). Thus, the genetic sex of the fetus is
determined by the haploid (23
chromosomes) genetic material of the
sperm. The presence of a Y chromosome
directs development of the male gonad, its
absence directs development of the female
gonad. This does not imply that the X
chromosome plays no role in sexual
development. The X chromosome contains
a number of genes essential to
development of both females and males.
For example, the gene encoding the
Figure 1. Sequence of events involved in sexual
differentiation.
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 Raul Martínez-Zaguilán, Ph.D.

androgen receptors is located

on the X chromosome.

2. Sex Differentiation.
The indifferent gonad anlage
forms at ca. 5 weeks of
gestation with the arrival of
the primordial germ cells
which migrate from their site
of origin near the yolk sac. In
the XY fetus, differentiation
of the testes is first evident at
ca. Week 6, with the
appearance of seminiferous
cords. The Y chromosome
exerts its effect on
development by controlling
expression of specific genes
of the sry (sex determining
region of the Y chromosome; Figure 2 Differentiation of human gonads. Testis development in the 8-
cf., Fig 1). The genes week. The sex cords lose contact with the cortical epithelium. The rete
expressed (formerly named testis develop in with 16 (cf., Fig 3). Ovary development in an 8-week
the H-Y antigen) are human embryo showing degeneration of primitive sex cords.
secreted by the Sertoli
precursors early in gestation. This causes
testicular differentiation (ca. Week 8; Fig
2). Gonadal cells in the ovary also
contain receptors for the H-Y antigen,
but since this protein is not produced in
females, these cells never receive the
signal for testicular formation, and the
indifferent gonad develops into an ovary
(Fig 3).

3. Differentiation of the Internal

and External Genitalia
Phenotypic sex refers to the structure of
the reproductive tracts and external
genitalia. Male genitalia (by week 16)
and female genitalia (weeks 11-20)
develop from the same common anlage; the wolffian duct. The 20-week human embryo does not
Figure 3 The rete testis develops at ca. week 16. The testis
cords are continuous with the rete testis and connect with
connect o the Wolffian duct , and new cortical sex cords
surround
2 the germ cells that have migrated into the genital
ridge.
 Raul Martínez-Zaguilán, Ph.D.

but this is not true for the reproductive tracts. The male reproductive tract is derived from
embryonic structures known as the wolffian ducts, while the female tract derives from the
müllerian ducts (Fig 4). The early embryo has the potential to develop either male or female
reproductive tracts because both duct systems are present before sexual differentiation occurs.

Development of phenotypic sex is determined by two hormones secreted by the fetal testis:
testosterone and müllerian inhibiting factor. Testosterone released from the Leydig cells stimulate
development of the wolffian ducts and differentiation of the male genitalia (weeks 8-16).
Müllerian inhibiting factor is produced by Sertoli cells and causes regression of the müllerian
ducts. Shortly after the fetal testis begins to form, both of these hormones are secreted.
consequently the mullerian ducts regress and the wolffian ducts and genitalia develop into the
male reproductive tract, penis and scrotum. Testosterone must be converted by 5 reductase to
DHT to cause virilization of the
external genitalia. This conversion
however, is not necessary for the
effects of testosterone on the wolffian
ducts. Therefore, both of these
androgens are required to provide
successful expression of the male
phenotype. Since there is no
testosterone released by the ovary in
the female, the wolffian ducts regress
and the external genitalia develops into
the clitoris, labia minora, and labia
majora (Fig 2). At the same time, in the
absence of müllerian inhibiting factor,
the müllerian ducts develop into the
oviducts, uterus and vagina.
Differentiation of the phenotypic sex in
the male occurs early in development
and is essentially complete by week 14- Figure 4 Differentiation of internal and external genitalia
16. During most of this period, there is
little or no secretion of LH by the fetal pituitary, so LH cannot be responsible for the critical
increase in testosterone production from the fetal testis. Instead, human chorionic gonadotropin
(hCG) from the placenta appears to be the stimulus for this early rise in testosterone secretion.

The relevance of the testes for proper development of internal genitalia has been demonstrated by
experiments in which only one testis was removed from embryonic rabbits (Fig 5). Under these
conditions, the müllerian duct regresses only on the side with the remaining gonad, indicating that
antimüllerian hormone mus act locally as a paracrine factor. The wolffian duct regressed on the

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 Raul Martínez-Zaguilán, Ph.D.

opposite side, suggesting that testosterone too must act locally to sustain the adjacent wolffian
duct because the amount that reached the
contralateral duct through the circulation were
inadequate to prevent its regression (cf., Fig
5).

4. Differentiation of the Nervous

System. In humans there are no inherent male
female differences in the ability of the
hypothalamus to secrete GnRH in response to
neuronal or hormonal stimulation.
Administration of estrogen to castrated male
monkeys or rats, for example, elicits release of
LH similar to those surges exhibited by
females.

However, important hypothalamic differences

must exist that explain the different sexual
Figure 5 Normal development of the male and
behavior observed between males and females.
female reproductive tracts. Shown in green are
These hypothalamic differences are established
tissues destined to be the male tract whereas those in
earlier during pre-natal or perinatal
purple are destined to be the female tract.
development, depending on the species.
Specifically, genetic female monkeys given testosterone during late fetal life manifest evidence of
male behaviors as adults (i.e., mounting behavior). It is therefore possible that similar brain and
hypothalamic differences exist in humans of different gender.

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 Raul Martínez-Zaguilán, Ph.D.

SUMMARY
________________________________________________________________
GONADS
________________________________________________________________
Gonadal Type Testis Ovary

Sex cords Medullary (internal) Cortical (external)

DUCTS
__________________________________________________________________
Remaining duct for Wolffian Müllerian
germ cells

Duct differentiation Vas deferens, epididymis, Oviduct, uterus, cervix

Seminal vesicle Upper portion of vagina
____________________________________________________________________