The effects of protein energy malnutrition (PEM) on sperm quality and spermatogenesis of male rats injected by testosterone enanthaie+)

Vol6, No I, January - March 1997

Effects of PEM on Sperm Quality and Spermatogenesis

The Effects of Protein Energy Malnutrition (PEM) on Sperm
euality and
Spermatogenesis of Male Rats Injected by Testosterone Enanthaie+)
Eliza*, N. Moeloek**, N. Suhana**, S. Sri Sukmaniah***

Abstrak
Telah diketahui, bahwa penyuntikan testosteron enantat (TE) kepada i
sampai sekarang belum diketahui apakah akibat penyuntikanTE terseùut dip

aenyebabiun aloopermia; namun
onon. Ooro

iùld ià'ùl|"rà

^4 (pada orang Kaukasia hanya
nya berbeda terhadap penyuntikanTE
ia menyebabkan 90Vo azoospermia), Diduga perbedaan tersebut

untuk mengetahui pengaruh diet rendah enersi protein

terhadap kualins sperma dan spermatogenesis tikus
inggu kepada tikus pEM akan menyebabkan penurunan kualitas
dalampenelitian ini adalah: konsentrasi sperma, morfologi normal
ormal maupun pada kelompok diet rendah kaloi protein; namun

menurun

matosonium-A dan jumtah spermatosit pakhiten juga
enersi protein, tetapi pada kelompok diet rendah enersi

::itHr::;;"!o"ii;i"::;:;

protein

mpok diet normal.

Abstract
The relationship of tesbsrerone enanthate (TE) with azoosDe

is also influenced by nutritionalfactor. Data of WHd andWaitisî

ns no difference

between the two groups. Testis weighr, diameter of the

the number ofA-spermatogonium, and the number ofpachyiene spermatocyte
were decreased

er TE injection; but there was a dffirence betvveen normal diet animals and pEM
animals,
Keywords: Protein energy malnutrion, Testosterone Enanrharc, Sperm
euality, Spermatogenesrs, Àars

Concern over the rapidly growing world's population

has stimulared andrologist to find effeciivè, safe,
and reversible contraceptive substances for men.
+) S_upported by The Project of Research Team URGE (University
Research For G raduate Educatio n) No. : 02 3/HTp p/U RGA I gg5.

Department of Education & Culture, Directorate General of Higher

*

Education.

Focutty

tf

Medicine, University

rf

An_

nt of Biomedical post Graduate program,
+* D.epartment of
Bioktgy, Facutty
donesia, Jukurta.

+** Depurment rf Nutrition,
Faculty
donesiu. Joktrta.

of Medicine, University of In_
of Medicine, University of [n_

Med J Indones

Eliza et al.

so that spermatogenesis can be induced' However,
protein dàficiency reduces the hypothalamus activity,
ihat in turn affecting the production of ABP'

mone secreted from hypothalamus, stimulates the syn-

thesis and release of

LH (luteinizing hormone)

and

This research is conducted to investigate the effect of
TE injection and malnutrition (protein energy malnutrifion = PEM) to sperm quality and spermatogenesis of albino male rats. It is hypothesized that
PEM male rats injected with 1 mg TE once a week for

(androgen binding protein) by the Sertoli cell of the
testis. Both testosterone and ABP have very important
role in the process of spermatogenesis' But the testosterone in high concentration decreases the production of LH and FSH directlY.
On the other hand, at the hyphophysis level hormone
with a direct influence to the process is more needed'Of
various hormone contained in contraceptive pills, combination of TE (testosterone enanthate) and DMPA
een shown
a or oligo-

evaluated.

hormones
of sperm,

tilitY' In addi-

normal
tion, th
membr

MATERIALS AND METHOD

gritY of sPerT
is hamPered'"

Animals used in this research were male albino rat
strain LMR (Lembaga Makanan Rakyat) (V/istar
derived). The number of experimental animals were
36, which were divided into two groups:

zoosPermia has
hether its effect

men response differently to TE.

It

one, consists of l8 animals and was given
food àn ad libitum base for up to three months old,
the animal weight of approximately between 150-

1. Group

.DataofWHO5
and Mongolian
has been suggested

200g(Normal=N)

that the different in their eating pattern might cause

n to TE

2.

(907o

(onlY 50% af-

Group two, consists of 18 animals and was given a

diet food to induce protein energy malnutrition;

therefore, their weights were lower, around6O-7OVo
of normal animals (correspond to protein energy

Bronson/,
ciencY in male

and

rats can reduce their hyphofisis activities. Consequent-

malnutrion = PEIvD.

Each group was divided into 3 subgroups:
- C (untreated control) animals (n = 6)
- TC (treated control = placebo control) animals (n =
6) injected with 0.2 ml solvent (wijen oil) once a
week begining at 12th week after birth.
- T (treated) animals (n = 6) injected with 1 mg TE
in 0.2 ml solvent (wijen oil) once a week begining
at 72th week after birth.

ly, the production of hypophysis controlled hormone,
zuch ai FSH and LH, which are important in the
spermatogenesis, will be disturbedAnother research showed that lower concentration of

Timetable

0 I 2 3 45 6 7 8 9l01ll2l3t4l5

161718192021

:=l=l==-:i::===:=-:

weeks afterbirth

normal food (group l)/diet food (group 2)
injected with TE or solvent
I

sacrificed

Vol 6, No

I, January - March

1997

Effects of PEM on Sperm Quality and Spermatogenesis

Animals were housed individually in 26x2lxll cm
cages and they were floored by saw-dust to absorb
urine. Testosterone enanthate (TE) and its solvent

(wijen oil) was made by PT Schering Indonesia. The
food was prepared in the Diponegoro unit of Nutrition
of the Ministry of Health Office. At the end of the
treatment (21 weeks afterbirth) all animals were

Sperm morphology (Normal head sperm)
Mean and SD of normal head sperm (%) of normal and
protein energy malnutrition (pEM) rats injected with
TE, is depicted in Figure 2.

sacrificed; both vas deferenses were excised and place

in petri dish containing 0.25 m1 0.85Vo NaCl. Both

testis were also excised, weighted and then fixed in
Bouin's solution then processed for histological

preparation and evaluation.

The parameters evaluated of sperm collected from yas

crcT cTcc
pachytene-spermatocyte. A Randomized Block
Design was used with two factors (Normal and pEM
animal) and three steps (injected by TE, solvent oil and
control). Data were normalized and homogenized
before statistical techniques were applied. Factorial
tests two by three were used to compare six different

treatments.
non-pa
variance (A
Test) were

mal,

then

is

of

cant
ele-

ments.

Figure 2.
C = untreated control; lÇ = placebo/treated control;
T = treated, injected with TE

Sperm viability
Mean and SD of sperm viability (Vo) of normal and
protein energy malnutrition (pEM) rats injected with
TE, is depicted in Figure 3.

RESULTS
The results of parameters evaluated are as follows:
Sperm concentration (vas deferens)

lion/ml) of
(PEM)rats
is depicted
80
60

tr
C TCT
N
Figure

lsr
2nd

CTCT
PEùI

L

C = untrealed control; TC - placebo/treatedcontro!:
T = treated, injectedwith TE

Figure 3.
C = untreated control; TC -- placebo/treated control;
T = treated, injected with TE.

Testis weight

Mean and SD of testis weight (mg) of normal and
protein energy malnutrition (pEM) rats injected with
TE, is depicted in Figure 4.

Med J Indones

Eliza et al.

250

1200
1000

2W

800

150

600

100

400
50

200

C TC T C TCT

Figure 6.

Figure 4.
C = untreated control;

lÇ

C = untreated control; lÇ = plncebo/treated control;
T = treated, injectedwithTE.

= plncebo/treatedcontrol;

T = treated, injectedwithTE.

A-Spermatogonium
Testis diameter
Mean and SD of testis diameter (cm) of normal and
protein energy malnutrition (PEM) rats injected with
TE, is depicted in Figure 5.

Mean and SD of A-spermatogonium number/tubule of
normal and protein energy malnutrition (PEM) rats
injected with TE, is depicted in Figure 7.

I

I

CTCT
N

CTCT
C TCT C TCT
PEM
N

PEI\,I

Figure 5.

Figure

Seminiferous tubule diameter

Pachytene spermatocYte

C = untreated control; TC -- placebo/treated control;
T = treated, injectedwithTE.

Mean and SD of seminiferous tubule diameter (pm) of
normal and protein energy malnutrition (PEM) rats
injected with TE, is depicted in Figure 6'

7.

C = untreated conlrol; TC - placebo/treated control;
T = treated, injected with TE.

Mean and SD of pachytene spermatocyte number/
tubule of normal and protein energy malnutrition
(PEM) rats injected with TE is depicted in Figure 8'

Vol 6, No 1, January - March 1997

Effects of PEM on Sperm

euatity and Spermatogenesis

the sperm concentration (p > 0.05) between TE
in_
jected PEM and TE injected normal
animals.
Sperm viability

C TCT C TCT
N
-E-'

ropped
al conas

Figure 8.
C = untreated control; TC placebo/treated
conlrol;
=
T = treated, injected with TE.

The Statistical Analysis

Testis weight

ANOVA tesr for testis weight showed that both in

Distribution and Homogeinity of data
Before analysis of variance (ANOVA) test was
carried

Testis diameter

Sperm concentration

S eminiferous

tubule diamete

A-spermatogonium
Sperm morphology

well

r

VoI6, No I, tanuary - March 1997

H uman R ec omb inan t Ery throp oi e tin

't6
14
12
10

I
6

4
2
0

2468101214
Weeks

30

Ht

Figure 4. Case no.4, male, 62 years old, Muttiple Myeloma
Hb

12

25

10

20

I

15

6

10

4

5

2

o

0

2468101214

16

Weeks

Figure 5. Case no. 5, male, 6g years old, Multiple Myeloma
40

Ht

Hb
a

a

14
12

30
10

I l;Hbl

20

. l*t,
4
2

4

6 ''= I

10

0
12

Weeks

Figure 6. Mean Hb, Ht,from 5 cases

14

I

43

Med J Indones

Eliza et al.

P achyt

ene

sP e

rmut o cY t e

(N)
ANOVA test showed that both in normal

and

malnutrition (PEM) animals TE injec-

p-Èin

"n"tgy
iion declineà- the number of pachytene spermatocyte
Gii'*'rry G < 0.01) compaied to tn" *ti,""ri$J.]

in

TE in-

al animals.

DISCUSSION
TE injecThe declining of sperm concentration after
maltion either on the normal (N) and protein energy
to
due
(PEM) animals is very likely' This is
directly
"",iiii".
t"g",it" t"edÉack of exogenous testosterone
"io-inïrtvpophysis which *pt"tt"t the production of
so
irr unaîsÉ or may be through the hypothalamus
the
,n" ftoat",ion of dH-lH decieases and eventually
of LH and FSH will also drop' In both

f.oâo"ti*

will be
cases, finally the process of spermatogenesis
of sperm consuppressed' Since there is no difference
between normal (N) and protein energy
centration

interaction
malnutrition (PEM) animals, there is no
energy-malnutrition
protein
and
injection
b",*""n TE
conon spermatogènesis, so the declining of sperm
cent;tion in this experiment is solely due to the TE
injection.
morThe same explanation is hold true also for sperm
sperm
normal
fnorogy, ,o ih" decreasing number-of.

TE injection that
-orpnôtogy was due to the cell
hampered IÉ" pro""tt of organel
.development'

normal
Since there was no difference the number of
normal
,f".- *otpttology between TE injected in both that
the
likely
very
pfur4
was
it
so
animats,
(fr) ana
to
due
number of sperm morphology was

à"ér"uring
the TE injection.

likely also the mechanism
the
were also found on the viability of the sperm in
declined
sense that the number of viable sperm also
animals;
PEM
and
normal
both
in
after TE injection

Thç

same results and very

this declining was also due to exogenous testosterone'
TE injection
because there was no tnteraction between
and PEM.

viability
The most interesting phenomenon is that the
also
animals
PEM
of sperm in the untiéated control
declined, although

in the treated control of PEM

due to the calory
disrupt the cell
be
deficiency; this deficience might
In
membrane with the end result affected the viability'
been
has
the treated control of PEM, this deficience
animals did

not. This is probably

than in normal (N) grouP.

The same explanation is hold true also for the number

VoI6, No 1, January - March 1997

Effects of PEM on Sperm Quality and Spermatogenesis

The histological preparations of seminiferous tubules
taken from normal (N) and pEM animals show the

difference of the rubules diameter (Fig. 9 and Fig.
10).

Figure 9' Histological preparation showing the seminiferous tubule of normal diet (N) animal; (C),
K
untreated control; Kp (CT),
control; T (P), injected by TE. Compare the smaller tubule diameter of treated animal.

treated,/placebo

tn

tr{n

I

CIO

Figure I0' Histological preparation showing the seminiferous rubule of protein
energy malnutrition (pEM) animal; K (C), uncontrol; KP (cT), lreated/placebo control; T (P), injected by T'8. compare the smaLler
tubul.e

treated

diameter of treated animal.

t0

Eliza et aL

Med J Indones

CONCLUSION
The effect of protein energy malnutrition (PEM) on
spenn quality and spermatogenesis of male rats in-

jected

l.

2.

with TE,

can be concluded as follow:

Sperm concentration, normal sperm morphology
and sperm viability was decreased, in both normal
diet and PEM animal; nevertheless there was no

difference between treated normal diet animals and
treated PEM animals.
Testis weight, diameter of the testis, diameter of the
seminiferous tubule, the number of spermato-

gonium-A, and the number of pachytene sper-

matocyte decreased in both normal diet and PEM
animal; but there was a difference between treated
normal diet animals and treated PEM animals.

of National Kongres I,
S

oci ety, Jakarta, I

9

Indonesian En-

86;22-229.

3. Frick J, Danner Ch, Kunit G et al. Spermatogenesis in men
treated with injection of mendroxyprogesterone acetate combined with testosterone enanthate. Int J Androl 1982;24652.

4. Moeloek

N.

deficiency on rat testicular function. Int J Androl 1980;

22:575-83.
9. French FS, Ritzen EM. Androgen binding protein (ABP) in

efferent duct fluid

of rat testis. J Reprod Ferfil.,

1973;

32:479-83.
10.

Nainggolan M. Experimental design (perencanaan dan
pengerjaan percobaan). GIS Research Institute, Medan
ta,1985.

Kontrasepsi pria masa kini dan masa akan
datang. Presented at The VI Yearly Scientific Meeting, Indonesian Andrological Society, Bandung, 1987.
2. Tadjudin MK. Cara keluarga berencana hormonal pada pria.
ocri nologi cal

tive development male and female mice and male rats.Am J
37 0-6.
8. Vawda AL, Mandlevana JG. The effect of dietary protein

Physiol 1986; 250:

Branch, Medan, Sumatra, 1965.
AD. Faktor gizi. Bharata Karya Aksara, Jakar-

l. Moeloek N.

Proceeding

989;3-516.
7. Hamilton GD, Bronson FH. Food restriction and reproduc-

I 1. Sediaoetama

REFERENCES

d

5. Word Health Organization. Contraceptive efficasy of testosterone induced azoospermia in normal men. Lancet,
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6. Waites GMH. The research strategy of the WHO task force
on methods for the regulation of male fertility. In: Perspectives in Andrology (Serio, M, ed), Raven Press, New York.

Penurunan kesuburan pria pada penyuntikan
DMPA dan l9 Nortestosteron heksiloksifenil propionat ( I 9-NT) + DMPA. Dissertation, Faculty of
Medicine University of Indonesia, Jakarta, 1991.
testosteron (TE) +

12. CIermontY. Quantitative analysis of spermatogenesis of the
rat : A Revised Model for the Renewal of Spermatogonia.
Am J Anat 1972:'lll: 111-29.
13. Franchimont P. Hormonal requalition of testicular function.
In : Regulation of Male Fertility. Cuningham GR, Sehell
WB, Hafez ESE (eds). Martinus Nifhoff Publisher, London.
1980;5-14.
14. Lermite V, Tergui M. Plasma sex steroid binding protein in
mature Heifers: Effect of the productive status, nutritional
level, and porcine growth hormone, and tradiol-l78 treatment. Bio Rep 1991; 44:864-70.
15. Street C, Herry RJS, Al-Othman S, Chard T. Inhibition of

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Med J Indones

Eliza et al.

P

lvent (wijen oil). Acordil contained 437o of WA

achyt en e s P ermat o cY t e

essential fattY acid which
mbrane integritY'

in normal (N) and PEM'
diameter
ini""tià'n caused decrease of weight and

Our finding showed that both

fe

oitt t"ttit

DISCUSSION

As
and also seminiferous tubule diameter'

can sup"
has been mentioned earlier, TE injection
feedback
through
Dressed the spermatogenesis
cell
It""ft*it*t acËordingly the number of germinal
is
it
So
decreased'
*irttin the tubules *itt utto
the
and
tubules
the
of
r"uto*tf" that the diameter
the testis
àiurn"t", of the testis and also the weight of
protein
factor'
à""i"ut"a. But for PEM group another
since
considered'
be
(PEMI should
^tl"utrion
""r-r1y
therJ was a difference between{E,iliiïîï"iffiï,:

grouP), for all three
s diameter and tubule
tatistical analYsis suPPort
interaction
such preposition, since there was positive
PEM'
betwËenlacbr TE injection and
14
reported that nutriermite and Tergui
tional status seem to be an additional factor regulating
binding protein (SBP) level which may

According to

T

ir*-tt"roU

injection.

the TE injection.

this declining was also due to exogenous testosterone'
TE injection
because there was no interaction between
and PEM.

of SBP available for positive or
in the
i"g",i* ieedback; the decline of SBP' results
in
serum'
androgen
concentration of free
inJreasine
'Stree*
eiat,l5 found that nonesterified fatty acids
to
modify binding affinities of sex steroid hormone
is
group
PEM
SBP t; vitro' Since in this experiment
acid
fatty
of
undernourish, it is predicted that supply
the
and protein is very limited; it follows by
the
if
over
avaitaUitity of SBP in the blood, more
the
blood
then
affinity to testosterone is also low,
alter the percentage

free téstosterone increases sharply upon the introduction of exogenous testosterone; followed by stronger
to norfeedback michanism in PEM group compared
will
induce
mal (N) group. Thus, this condition
,t ong"i ùppàssion of spermatogenesis in PEM
than in normal (N) grouP.
The same explanation is hold true also for the number
so
of A-spermatogonim and pachytene spermatocyte'
were
cells
germinal
the
the decreasing number of both
due to the TE injection that hampered the proliferation
during spermatogenests. Since there was a difference
U"t*J"tt fg injected in normal (N) and PEM animals
very
concerning to the number of germinal cells' it was
a
atogoni
A-sperm
of
g number
I ikely thaithe decreasin
due
was
animals
and iachytene spermatotyte in PEM

to the interaction between TE injection and PEM'