The influence of immobilization stress on spermiogenesis in rats was investigated. After 96 hour immobilization, histological changes began to manifest themselves in the form of practically complete disappearance of cell population of the wall of seminiferous tubule as well as a markedly increased number of cells with pathologic mitoses. Enzymological investigations showed various changes of activity (of acid and alkaline phosphatase and nonspecific esterase) in the 24, 48, and 96 hour immobilization groups

Meitner, E. R.

English

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NASA TM-76282
EFFECTS Or 1VIMOBILIZATTON ON SPERMIOGENESIS
E. R. Meitner
Translation of "E nfluss der Immobilisation auf die Spermiogenese,"
Acta Anatomica, No. 95, pp 300-308y, 1976
(NASA -TM- 76282) EFFECTS OF IMMO.B.IL;IZ.A11ON	 N80-27993
ON SPERMSOGEN'ESIS (National Aeronautics and
Space Administratiou) 14 p HC A02/MF A01
C ,C:L 06C	 Unclaa
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NATIONAL AERONAUTICS AND SPACE ADMINISTRATION
WASHINGTON, D.C. 	 ,TUNE 1980
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EFFECTS OF IMMOBILIZATION ON SPERMIO-
 GENESIS
E. R. a^dEYTNER	 € F'ertori.. ,ng Qrgonrzor.tn spurt No.
1
f	 ff-t.;r,r.rg ^+ a^an.sat,n^ Nome ar,r+ Address
	 -
Teo Kanner Associates
Redwood City, California 94063
^'	 " Fr "xo = nn A ;sncy Name and A,1 3res'
National Aeronautico and Space Admin-
istration, Washington, D.C. 20546
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Supplementary Notes ........_...,^^.,..,.,,.,....^.^.,e.......^., ....^.,..^,..,^.^,.,.,a^...
Translation of "Einfluss der Immobilisation auf die Spermio-
genese, " Acta Anatomica, No. 95, pp 300-308 / `f7 L,
a
16. Abstract
The author investigated the influence of immobilization
stress on spermiogenesis in rats. After 96 hour immobili-
zation, histological chan ges began to manifest themselves
in the form of a practically coi-tiplete disappearance of cell
population of the wall of seminiferous tubule as well as a
markedly increased number of cells with pathologic mitoses.
Enzymological investigations have shown in particular group,
(24 48 and 96 hour immobilization) various changes of ac-
tivity (of acid and alkaline phosphataae, nonspecific ester-
ase) which, after temporary negativity, became positive
again. in the last group.
	
t1i: P s	 1 " 3 1- 1 ,	 TY1
17, Key Words (Salected by Author(s))
	 19, Distribution Statement
Rat. Spermatogenesis.
Immobilization. Cell
destruction. Pathological 	 Unlimited-Unclassified
mitoses. Phosphatases.
19. Security Clossif, to( this report)	 t 24. $ocurity Ciassif, (of this page;
	
Zl• No. of Pages	 22. fries
Unclassified	 Unclassified	 12
^,1D Work Unit No.
11. Contract or Grant No.NASw- _31_9 9 _
13._7Vp'"
 of deport and p eriod Covered
Translation
14. $ponsoring Agency Code
EFFECTS OF IMMOBILIZATION ON SPERMIOGENESIS
E- R- MEITNER
Chair of Histology and Embryology, Medical Faculty of the
Comenius University, Martin, CSSR
Introduction
	 300*
The effects of temperature, light, various infectious diseases
and of cryptorchism on the germinal epithelium of the testes are well
known. They involve specific morphological and functional changes.
We became interested in determining whether general changes in the
state of the entire organisx;i due to immobilization stress would have
any effects of spermiogenesis.
Materiel and Methods
In our experiments we used nine adult Wstar rats (three other
rats served as control animals) divided in groups of three animals
each, which were immobilized for 24, 48 and 96 hours individually in
such a manner as to prevent any outside effects on their testes. The
testes removed immediately following decapitation were subjected in
part to HE-staining (paraffin sections), partly to enzymatic reactions
(Baker, frozen sections) .
Description of Findings	 301
In normal state the seminiferous tubules show the layer arrange-
ment of spermiogenesis, from spermatospores over spermiocytes and
spermatides to sperm filling the lumina of the tubules (111. 1). Fol-
lowing a 24-hour immobilization, no pronounced changes can be detected
'Numbers in the margin indicate pagination in the foreign text. 	
1
in the cell population of the Tubuli seminiferi: the layer sequence of
germ cell generation is undisturbed. Following a 48-hour immobiliza-
tion the numk;,r
 of spermatospores apparently decreases, spermiocytes
have lost some of their size and chromatin of spermiocytes of the 2nd
order have somewhat weaker coloring. Numerous spermatides die off;
the volume of the seminiferous tubules contains only isolated sperms
the cell nuclei of which have no coloring (Ill. 2). Filamentous crys-
talloids appear in varying quantities among the spermiogenetic cells.
The greatest changes were detected following a 96-hour immobilization.
The number of ,spermatospores with hyperchromic nuclei showed a consid-
erable decrease. Regeneration of all cell types of spermiogenesis
leads in numerous sections of the seminiferous tubules to complete
disorder in the layer arrangement; in many cases the wall of a semini-
ferous tubule consists only of loosely suspended undeveloped seminal
cells. In the aften only isolatedly in the lumen sporadically present
spermiocytes with highly prismatic cell plasma, which are sometimes
mutually connected by cytopl-em.ic bridges, appear indications of nuclei
disintegration: the cytoplasma of these cells shows a honeycomblike
structure, and in all cases also small vacuoles, as signs of necro-
biosis. Spermatides and sperms occur only sporadically and in very
small numbers. Sperm agglutination is never detected in the lumina
of the tubules, all that can be seen there are nuclei of dead cells.
The overall picture gives the impression of a total decomposition of
the spermatogenic cell population (Ill.. 3). Numerous spermatospores
are converted into small, oval, hyperchromic elements without detect-
able mitosis modes. It is often possible to find on the Membrana
propria tighly attached ovoid nuclei poor in chromatin content with
a dark plasmosome which resemble Sertoli's cells even though their
I'l
2
Longitudinal axis is parallel to the Tunica propria. In some spots
they occur in larger quantities and crowd spermatospores away from the
Membr, propria (Ill. 4). Presence of eosinophilous liquid in tubule
sections is not a rare occurrence (111. 5).
A very remarkable and frequent change consists in appearance of
large, round to polyhedral cell elements with varying pathological
mitosis modes and pale cytoplasms which often occur in multiple Layers 304
in the entire circumference of the tubule, sometimes isolated in the
Lumen, only with difficulty identifiable as a type of spermiogenesi.s
cells (111. 6). These isolated cells are found on the inner surface
of the Membrana propria and, thus, must be interpreted as undeveloped
seminal cells -- in spite of pathological m.i.toschisis. In several
cases we found nuclei modifications resembling an eccentrically situ-
ated hollow nucleus (111. 7). In other loci we detected in the cells
one or two vacuoles in addition to pathological mitoses. Between
cells with pathological mitosis modes there is a frequent occurrence
of plasma residues of dead cells, vacuolated cells and filamentous
crystalloids (111. 8). Elsewhere we found spermiocyte chromatin --
as an indication of nucleus pyknosis -- agglutinated into two homo-
genous dark-blue clumps.
For enzyme-histochemical studies we performed the following re-
actions: 1. Gomori's reaction to acid phosphatase, 2. Naphthol-AS-Bx-
phosphate (pH 5.5) to acid phosphatase, 3. c*ap latholphosphate (pH 5.5)
to acrid phosphatase, 4. a/naphtholphosphate (pH 9.2) to alkali: phos-
phatase, 5. Reaction to unspecific esterases and 6. Reaction to glu-
cose-6-phosphatase.
3
The following findings were made on control ar ►imals ; 1. acid
phosphatase according to Gomori shows in the central ,arts of the wall
of the seminiferous tubules activity in the form of a brown precipi-
tation (111. 9); 2. and 3. In both reactions the acid phospha-Laze
showed activity in peripheral sections of the wall of the seminiferous
tubules in the form of a reddish or red-brown coloring; 4. The naph-
tholphosphate reaction to alkalis phosphatase took the form of a fine,
very thick, black, granular precipitation active in the peripheral
Tubulus section (111. 10); 5. The non-specific esterases were active
throughout the width of the wall in the form of blue grains up to	 3.06
striate granular agglutinations radially converging on the lumen of
the tubule (111. 11); 6. The glucose-6-phosphatase is active in the
central sections of the seminiferous tubules in the form of coarsely
granular brownish precipitation (Ill. 12).
The enzymes studied in the walls of seminiferous tubules showed
the following changes after a 24-hour immobilization; Reaction sub 1
was more intensive in most of the tubuli (Ill. 13). Reactions sub 2
and 3 were for all practical purposes negative, and the reaction sub 4
was completely negative. The reaction listed sub 5 showed a very
greatly diminished activity (111. 14), and reaction sub 6 was also
diminished. The 14-hour immobilization: led thus to an increased
activity of acid phosphatase (according to Gomori), the reactions to
acid phosphatase (naphthol-AS-DI,a-naphthol) and the reaction to al-
kalic phosphatase (a-naphthol) were negative, reaction to non-specific
esterases was strongly diminished, as was the activity of glucose-6-
phosphatase.
4
Following a 48 -hour immobilization, the reaction sub 1 was strong-
ly positive (111. 15), reactions sub 2, 3, 5 (Ill. 16) and 6 (111. 17)
were positive, while the reaction sub 4 was negative.
Following a 96-hour immobilization, activity of acid phosphatase
(according to Gomori) was considerably stronger than in the control
animals (111. 18). Reactions slo 2, 3, 4 (111. 19) and 5 (111. 20)
were positive, while glucose-6-phosphatase showed no activity.
In summary it can be stated that immobilization has varying ef-
fects on the activity of the studied enzymes. With the exception of
acid phosphatase (according to Gomori) and the glucose-6-phosphatase
which snow activity after 24 and 48 hours, activity in the case of
acid and a.lkalic phosphatase and of non-speoific esterases is negative.
It is noteworthy that followingf a 96-hour immobilization the activity
of acid phosphatase (according to Gomori) exceeds that of 'the control
animals and that other enzymes, with the exception of glucose-6-phos-
phatase, show signs of activity. Even taking .nto account the indi-
vidual immobilization groups ( 24, 48 and 95 hours) , the behavior of
enzume activity varies. After .initial negativity it again becomes
positive.
Discussion
The described changes in the cell population of seminif erous tu-
bules are most pronounced following a 96-hour immobilization. We find
two morphological states. In part there occurs a complete restoration 307
of the spermiogenic layers of the tubules with a practically empty
lumen, in part there occurs the formation of numerous irregularly
distributed large cell elements with pathological mitoses. All that
5
can be identified in the surfaces of tubule walls built up of such
cells are hyperchromic spermatospores without witosis modes. Between
the large pale cells are crystalloids that occur also during semini-
f erous tubule changes due to other etiology; plasma residue of dead,
cells is found in the tubule's lumen. Both changes in the cell pup-
ulation, regressive as well as proliferative, during which there oc-
curs a complete suspension of spermiogenesis, we choose to interpret
as the result of negative effects on spermiogenesis by the 96-:hour
immobilization, in all layers of the cell population of the semini-
ferous tubules, whereby formation of cells with pathological mitoses
can be viewed as the effort of the organism to keep up even under a
spermiogenic stress situation -- even though in a roundabout way and
without attaining the final goal, i. e., without 	 formation of good
ripe sperm.
We refrain from making any statements regarding the specific type
and manner in which the described changes occur in seminiferous tubules
through immobilization. We just generally assume that the complex ef-
fects of the immobilization state which adversely influence metabolism,
the hormonal balance, blood circulation, the neurovegetative and acido
basic balance, are responsible for the micromorphological and enzyme-
histological changes. The fact that germinal epithelium reacts to
negative effects with regenerative, and in our experimental arrange-
ment also with cell proliferation changes, points to the easy vulner-
ability (also through nonmechanical effects) of the testes.
The regenerative changes occurring in 96-hour immobilization re-
semble those occuring in diphtheria and in cryptorchism. Comparable
or similar changes can be caused in the testes by serious psychical
6
traumata (STIEVE, 1925, 1930), as well as by pharmaceutical prepara-
tions with antiandrogenic effects (G,yproteron acetate - ,Androcur:
NEUMANN, 1973). Chemical and hormonic effects (Lysergamid or Corti-
son, thyroxine, among others) on spermiogenesis were also described
(MELLAN et al, 1968; GO et a1, 1971). Fasophilic crystalloids are in-
terpreted by STIEVE (1930) as regenerative phenomena in the testes of
older men. As shown by our experiments, they can also occur in general
stress situations. In connection with our experiments, we found no
308
references in available literature to formation of cells with patho-
logical. mitoses.
The results of micromorphological and enzymological studies con-
firm an adversary behavior between cytological and enzymatic changes.
Even though the wall structure of seminiferous tubules is completely
upset following a 96-hour immobilization, In other areas the activity
of acid phosphatase exceeds that of the control, animals and also that
of other enzymes will become positive after a temporary negativity.
The general state of the organism adversely affected by immobilization
progressed differently in the individual groups in relation to enzyme
activity of the seminiferous tubules' population,.
Summary
The author investigated the influence of immobilization stress
on spermiogenesis in rats. After 96-hour immobilization, histological
changes began to manifest themselves in the form of a practically com-
plete disappearance or the cell population of the wall of seminiferous
tubule as well as a markedly increased number of cells with pathologic
mitoses. Enzymological investigations have shown in particular groups
7
(24, 48 and 96-hours immobilization) various changes of activity (of
acid and alkaline phosphatas+e, nonspecific esterase and glucose-6-
phosphatase) which, after temporary negativity, became positive again
in the last group.
8
m .
/302
Its
%3
Effects of Immobilization on Spermio(Tenf:sis	 / 30 ^
V
9
UC`r	 Effecta of Immobilization on Spermiorrenesis
^4 }.
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i
ak
CAPTIONS TO ILLUSTRATIONS:
Ill. 1. Normal seminiferous tubules of an adult rat. HE, x64. 13o2
Ill. 2. Seminiferous tubules followinn 48-hour immobilization:
reduced number of spermatospores, dead spermatides, isolated sperms
without stained cell nuclei. IIE. x64.
Ill. 3. Seminiferous tubules following 96-dour immobilization:
dissolution of the spermatogenic cell population. tfE. x 64.
Ill. 4. Wall of a seminiferous tubule following 96-1 ►our immobi-
lization: multiplied Sertoli cells with low chromatin content prolif-
erating on the Membrana propria. ILE. x 640.
Ill. 5. Seminiferous tubules following 96-Hour immobilization:
eosinopphilous liquid in lumen. IT, xG4.
I11. 6. Seminiferous tubules following 96-Hour immobilization:
numerous pathological mitosis modes. 1IE. x160.
10
111. 7. Hollow nucleic call elements in saminifarous tubules LLOUfollowing 96-hour immobilization. M x640.
111. S. Pathological i4itoses plasma residue, crystalloids in
seminiferous tubules foll wing 96-hour immobilization. HE. x640.
111. 9. Control animal: acid phosphatase (Gomori) active in the
central parts of the sominifarous tubule wall. x64.
111. 10. Control animal.: alkaline phosphatase (a-naphtholphos
phate reaction), activity in peripheral tubulus section. xl6p.
11.1. Ill Control animal.. activity of nonspecific esterasoa
throughout the wall width. x64.
111. 12. Control animal: activity of glucose-6-phosphatase in
central section of the seminiferous tubule. x64.
111. 13. 24-hour immobilization, intensive activity of acid	 ILOUphosphatase. x64.111. 14. 24-Lour immobilization, reduced activity of nonspecific
esteras+es. x64.
111, 15. 48-hour immobilization, intensive activity of acid
phosphatass. x64.
111. 16. 48-hour immobilization, weak positivity of nonspecific
esterases. x64.
111. 17. 48-hour immobilization, pasivity of glucose-6-phosphat-
ass. x64.
111. 18. 96-hour immobilization, strongly intensive activity of
acid phosphatas e. x64.
111. 19. 96-hour immobilization, positivity of alkaline phosphat-
ass. x64.
111. 20. 96-hour immobilization, positivity of nonspecific ester-
asss. x64.
11
REFERENCES
Go, Q.L.W. et al., "Hormoneller Einfluss auf die Spermiogenese," (Hor-
monic Effects on Spermiogenesis), Can. J. - Bioch m. ,, 9, pp 768-775,
1971.
Mellan, J. et al. , "Chemischer Einfluss auf die Spermiogenese, " (Chem-
ical Effects on Spermiogenesis ), Activitas , nnerv.sun. , 10, pp.
274-275, 1969.
Neumann, F. "Cyproteronacetat -- tierexperimentelle Grundlagen,"
(Cyproteron Acetate -- Application in Animal Experiments),
Medsche Mitt, Scherincx, 2, pp. 2-10, 1973.
Stieve, H., "Samezellenverklumpung (Spermaag lutination), niche
Spermiopllagie " (Seminal Cell Clumping (Sperm Agglutination), not
Spermi.ophagia^, Z. mikrosk.-anat. Forsch., 2, pp. 598-629, 1925.
Stieve, H., In von Moellendorff's "Handbuch der mikrosko ischen
Anatomie des Monschen," (Handboo o . : uman croscopi.c natomy),
Vol. 
	 pr nger, Berlin, 1930.
Docent E. R. MEITNER.,, M.D. , Cand. Sci. , Chair for Histolo gy and Embryo-
logy, Medical Faculty, 6 Museum Street, 036 01 Martin 1 (CSSR)
12


Effects of immobilization on spermiogenesis

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