Ultrastructural Deficiency in Autonomic Innervation in Cremasteric
Muscle of Boys With Undescended Testis
By F. Cahit Tanyel, Sevda Muftuoglu, Attila Dagdeviren, Lale Karakoc, and Nebil Buyukpamukcu
Ankara, Turkey
Background/Purpose: The cremaster muscles (CM) associ-
ated with undescended testis reveal neurogenic alterations
that mainly affect type 2 fibers. The ultrastructure of CM has
been evaluated to define if further evidence to explain the
alterations could be identified.
Methods: CM of 8 boys with inguinal hernia and 8 boys with
undescended testis at similar ages were biopsied. Samples
were processed for electron microscopic evaluations. Semi-
thin and thin sections were examined under an electron
microscope.
Results: The CM associated with inguinal hernia showed
normal ultrastructure. However, some alterations were en-
countered in CM associated with undescended testis. Unmy-
elinated fibers were diminished in number, and myelinated
fibers were outnumbering the unmyelinated fibers. Marked
disorientation of myofibers, redundant sarcolemma, empty
sleeves of basal lamina, disarray of myofibrils, densely
T HE GUBERNACULUM recently has been pro-
posed to represent the primitive mesenchymal tis-
sue that precedes the myogenesis.1 Gubernaculum ceases
to exist after giving rise to smooth muscle, which, at the
periphery, subsequently transdifferentiates into the cre-
master muscle (CM).2 The physical force to descend the
testis has been described to be the coordinated propulsive
activity of the smooth muscle and CM.1,3 Smooth muscle
has been defined to be a transient structure, which should
disappear through apoptosis after the descent.4 Although
an alteration in innervation has been suggested to impair
the propulsion, thus, hinder the descent of testis,3,5,6
persisting signals to the smooth muscle has been pro-
posed to inhibit the apoptotic process and result in
inguinal hernia or hydrocele depending on the rescued
amount of smooth muscle.7,8
Pharmacologic and histopathologic evaluations of CM
associated with undescended testis have shown alterations
in contractile properties and a neurogenic damage possibly
reflecting the absence of b-2 adrenergic effect.5,9-12
Because the affected CM appears to have a role in the
failed descent, the precise definition of alterations in CM
associated with undescended testis may provide addi-
tional evidence to enlighten the reason for failed descent.
Therefore, the ultrastructure of CM associated with un-
descended testis was evaluated and compared with those
associated with descended testis but inguinal hernia.
Journal of Pediatric Surgery, Vol 36, No 4 (April), 2001: pp 573-578
packed myofilaments, Z disk streaming, dilated sarcoplasmic
reticulum, and dense-irregularly shaped mitochondria were
repeatedly encountered. Satellite cells appeared inactive.
Most of the fibers were contracted.
Conclusions: The decrease in number of unmyelinated fibers
appears to represent a decrease in autonomic nerve fibers.
The alterations within muscle fibers may reflect a deficiency
in autonomic innervation. Autonomic nervous system is
highly responsive to circulating androgens. Factors decreas-
ing the vulnerability of autonomic nervous system against
androgenic effects may result in a CM with neurogenic alter-
ations, thus inhibiting testicular descent.
J Pediatr Surg 36:573-578. Copyright 2001 by W.B.
Saunders Company.
INDEX WORDS: Electron microscopy, cremaster muscle,
cryptorchidism, autonomic nervous system, androgen.
MATERIALS AND METHODS
CM were obtained from boys with inguinal hernia (n 5 8) or
undescended testis (n 5 8). Tissue samples were first fixed by immers-
ing in 2.5% gluteraldehyde in phosphate buffer and postfixed in 1%
osmium tetroxide in phosphate buffer. After dehydrating in ethanol
gradients at room temperature, the samples were embedded in araldite
(araldite CY 212, TAAB). Semithin (1-mm thick) and thin (70-nm
thick) sections were cut. Semithin sections were stained with toluidine
blue Azur II, and thin sections were stained with uranyl acetate and lead
citrate. The specimens were examined and photographed under electron
microscope (Zeiss EM9-S2; Karl-Zeiss, Oberkochen, Germany).
RESULTS
The ages of boys with undescended testis or inguinal
hernia have been similar (32 6 6 and 30 6 8 months;
P . .05).
Findings in Samples Associated With Inguinal Hernia
The nerve fibers, myofibers, and associated organelles
in CM from boys with inguinal hernia have shared
From the Departments of Pediatric Surgery and Histology-Embry-
ology, Hacettepe University, Faculty of Medicine, Ankara, Turkey.
Address reprint requests to F. Cahit Tanyel, MD, Hacettepe Univer-
sity, Childrens Hospital, Department of Pediatric Surgery, 06100
Ankara, Turkey.
Copyright 2001 by W.B. Saunders Company
0022-3468/01/3604-0008$35.00/0
doi:10.1053/jpsu.2001.22285
573
574 TANYEL ET AL

ultrastructural properties common to striated muscles

(Figs 1 and 2).

Findings in Samples Associated With

Undescended Testis
The connective tissue around the myofibrils contained
several nerve fibers. The perineural sheaths of the nerves
were normal. Endoneurium contained numerous bundles
of collagen fibers and connective tissue elements. Un-
myelinated fibers were diminished in number, and my-
elinated fibers were outnumbering the unmyelinated fi-
bers (Fig 3). Schwann cells and the axons enclosed by
them almost were normal. Besides normal-appearing
myelin sheaths, myelin sheaths with decreased thick-
nesses were encountered. Some of the myelin sheaths
have shown indentations and loop formations. Although
a typical motor end plate was not encountered during
evaluations, both myelinated and unmyelinated nerve
fibers close to the striated muscle fibers were observed.
The axon terminals were rich with mitochondria and
synaptic vesicles.
The myofibrils were both smaller and irregular com-
Fig 2. The same muscle through higher magnification (3 88,000,
pared with those associated with inguinal hernia. Disar-
uranyl acetate-lead citrate).
ray of myofibrils, Z disk streaming, dilated sarcoplasmic
reticulum, and dense, irregularly shaped mitochondria fibrils (Fig 4). In addition to the marked disorientation of
were encountered repeatedly. The muscle fibers have had myofibrils, empty sleeves of basal lamina projecting
large spaces at the peripheral sarcoplasm that lack myo- from the surface were encountered. Myonuclei were
normal. However, myofilaments were packed densely,
and sarcolemmas were redundant (Fig 5). Besides pe-
ripheral spaces, halos between myofibrils also were ob-
served. The halos around the myofibril clumps contained

Fig 3. A nerve fiber in connective tissue between the muscle

fibers. The perineural sheath has normal structure, but the endo-
Fig 1. The regularly oriented myofibrils with the Z lines, A and I neurium is rich in collagen fibers, but poor in myelinated (thick
bands in a cremaster muscle associated with inguinal hernia (original arrow) and nonmyelinated axons (double thin arrows). (Original
magnification 3 11,000, uranyl acetate-lead citrate). magnification 35,800, uranyl acetate-lead citrate.)
ULTRASTRUCTURAL DEFICIENCY IN UNDESCENDED TESTIS
Fig 4. Peripheral areas devoid of myofibrils, but containing gran-
ular material (original magnification 3 4,750, uranyl acetate-lead
citrate).
dense, irregularly shaped mitochondria and dilated cis-
terns of sarcoplasmic reticulum (Figs 6 and 7). Most of
the muscle fibers appeared to be contracted because
H-zones of each A-bands have been narrow and difficult
to differentiate. Mitochondria have appeared as small,
irregular, electron-dense structures, with the half size of
those encountered in samples from boys with inguinal
hernia.
Fig 5. Two adjacent muscle fibers with redundant sarcolemma
and densely packed myofilaments that are in accordance with a
denervated fiber. The fibers have empty sleeves of sarcolemma
(asterisks) and markedly disoriented myofibrils (original magnifica-
tion 3 56,000, uranyl acetate-lead citrate).
575
Fig 6. Empty spaces between myofibers, electron dense, and
irregularly shaped mitochondria of different sizes (double thin ar-
rows), and enlarged cisterns of sarcoplasmic reticulum (asterisk).
(Original magnification 3 56,000, uranyl acetate-lead citrate.)
Small satellite cells that have appeared to be inactive
through silent cytoplasm and resting heterochromatic
nucleus were observed.
DISCUSSION
Although the CM is known to develop within the
gubernaculum,13,14 and the gubernaculum is accepted to
be the pivotal structure for testicular descent, CM is
poorly evaluated in humans. The ultrastructure of the
Fig 7. Dilated sarcoplasmic reticulum between myofibrils (aster-
isk). (Original magnification 3 120,000, uranyl acetate-lead citrate.)
576
CM in humans with either descended or undescended
testes is unknown.
Nerves to muscles contain both myelinated and unmy-
elinated axons. Although most of myelinated fibers rep-
resent the motor fibers, some of the unmyelinated fibers
represent autonomic efferent fibers. However, afferent
fibers from muscle spindles, Pacinian corpuscles, smaller
paciniform, and unencapsulated free endings are myelin-
ated, and those terminating as free endings in the inter-
stitial connective tissues are unmyelinated. Unmyeli-
nated fibers are the most numerous component of the
afferent system that contributes to two thirds of the total
sensory innervation.17 In a peripheral nerve, the unmy-
elinated fibers outnumber the myelinated fibers.18 The
decrease in number of unmyelinated fibers with an ex-
cess of myelinated fibers suggests that autonomic or
unmyelinated afferent fibers are decreased in CM asso-
ciated with undescended testis. The clinical conditions,
which cause a decrease in both sensory and autonomic
fibers, such as hereditary sensory and autonomic neurop-
athy, are well known entities.19 In those clinical condi-
tions, involvement of the afferent system also causes
alterations in electromyography findings. Electromyo-
graphy of the cremaster reflex, which has not shown a
prolonged conduction velocity, does not suggest the
involvement of either the afferent system or the motor
neurons in boys with undescended testis.6 Therefore, the
decrease in number of unmyelinated nerve fibers in the
current study appears to represent a deficiency in auto-
nomic innervation.
Some of the alterations encountered in the muscle
fibers such as redundant sarcolemma, densely packed
myofilaments, marked streaming of Z discs,20 small mi-
tochondria with an increase in electron density,21 dilated
sarcoplasmic reticulum, and normal myonucleus, halos
around the myofibril clumps have been similar to those
encountered after denervation.22 However, the inactive
appearance of satellite cells differs from those that ap-
pear active after motor denervation.23
Innervation is required for latter stages of myogen-
esis,15 and the phenotype of muscle depends on the
nerve.16 In addition to the unaffected motor nerve
through electromyography,5 normal transformation of
type 2 muscle fibers to type 1, which reflects the matu-
ration governed by motor innervation,24 is not affected in
CM of children with undescended testis.11,12 The ultra-
structural alteration related to a defective autonomic
innervation of a striated muscle is not evaluated in the
literature. Catecholamines influence metabolism and ex-
citability of mammalian skeletal muscles.25,26 These ef-
fects are mediated through beta adrenergic receptors.25
Skeletal muscle beta adrenergic receptors are shown to
be predominantly b-2 subtype.26 Beta-2 adrenergic re-
ceptors also have been shown in the CM of the guinea
TANYEL ET AL
Fig 8. The proposed pathway to explain the ultrastructural
alterations in cremaster muscles associated with undescended
testis.
pig.27 The stimulation of those receptors additionally
increase the sarcoplasmic cAMP and activate calcium
pumping by the sarcoplasmic reticulum, which in turn
decreases intracellular Ca21.28 Lack of b-2 adrenergic
effect might be associated with a disturbed energy me-
tabolism and higher levels of intracellular Ca21. Because
our findings cannot be explained by denervation, and the
peripheral nerve contains fewer unmyelinated fibers, we
consider the alterations within myofibers to reflect those
resulting from a decrease in autonomic innervation. The
decrease in b-2 adrenergic effect also may explain the
more profound alterations in type 2 muscle fibers,12 as
well as findings that suggest a calcium overload such as
contracted fibers encountered in the current study, and
increased amplitude of tetanic contractions of CM asso-
ciated with undescended testis.5
The genitofemoral nerve is known to be important for
the descent of testis.29 The reason that causes a decrease
in the number of autonomic neurons within the genito-
femoral nerve that innervates the CM appears to be
responsible for the failed descent.
Neurons initially are overproduced. Approximately
one-half of the original population is lost through
programmed cell death. Neuronal survival is con-
trolled by neurotrophic factors and genes that control
the cell death cascade.30 Sympathetic ganglia are vir-
tually absent in mice carrying mutations of nerve
growth factor or trk A genes. In addition, mice lacking
the neurotrophin 3 gene have a reduced number of
sympathetic neurons.31 However, autonomic neurons
are reported to survive without target derived tro-
phic factors. Typical developmental cell death that
affects motor neurons through target dependency has
been questioned for autonomic neurons,32 and their
development has been proposed to be under central
control.33
Another factor important for the descent of testis is
androgen.34 According a widely accepted theory, the
androgens affect the descent via the genitofemoral
nerve.35 Alterations within both the motor nucleus and
the genitofemoral nerve itself associated with antiandro-
ULTRASTRUCTURAL DEFICIENCY IN UNDESCENDED TESTIS
gen-induced undescended testis support the view that
androgenic effects to descend the testis involves the
peripheral nerve.36,37 Because the motor nucleus of the
genitofemoral nerve is sexually dimorphic, the andro-
gens have been considered to influence the descent via
the motor unit. However, the autonomic nervous system
also is sexually dimorphic.38 Although the cholinergic
neurons are less sensitive to androgens, noradrenergic
neurons are dramatically affected from circulating andro-
gens.39 Many components of pelvic autonomic reflex
pathways, including preganglionic neurons, autonomic
ganglion cells, and primary afferent neurons, are influ-
enced by testosterone. Although the mechanism of action
currently is unknown, androgens obviously are important
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