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

_{aenyebabiun aloopermia; namun} sampai sekarang belum diketahui apakah akibat penyuntikanTE terseùut

dip

^4 onon. Ooro

iùld ià'ùl|"rà

nya berbeda terhadap _{penyuntikanTE (pada orang Kaukasia hanya} 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 matosonium-A dan jumtah spermatosit pakhiten juga

menurun

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

enersi _{protein, tetapi pada kelompok diet rendah} enersi 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 of Medicine, University of In_

donesia, Jukurta.

Eliza et al. Med J Indones

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

This research is conducted to investigate the effect of TE injection and malnutrition (protein energy

mal-nutrifion

=

PEM)

to

sperm

quality

and

sper-matogenesis of albino male rats. It is hypothesized that PEM male rats injected with 1 mg TE once a week for

evaluated.

MATERIALS AND METHOD

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:

1.

Group 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

150-200g(Normal=N)

2.

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 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.

weeks afterbirth

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

mone secreted from hypothalamus, stimulates the syn-thesis and release

of

LH

(luteinizing hormone) and

(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 tes-tosterone in high concentration decreases the produc-tion 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, com-bination

of

TE (testosterone enanthate) and DMPA een shown a or oligo-hormones

of

sperm,

normal

tilitY' In

addi-tion,

th

gritY of sPerT

membr

is hamPered'"

zoosPermia has hether its effect .DataofWHO5 and Mongolian men response differently to TE. It has been suggested that the different

in

their eating pattern might cause

n

to TE (907o (onlY 50% af-Bronson/, and ciencY in male rats can reduce their hyphofisis activities. Consequent-ly, the production of hypophysis controlled hormone, zuch

ai

FSH and

LH,

which are important

in

the spermatogenesis, will be

disturbed-Another research showed that lower concentration of

Timetable

0 I 2 3 45 6 7 8 9l01ll2l3t4l5

161718192021

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

Vol 6, No I, January - March 1997

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 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

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.

_mal,

then

non-pa

_{is of}

variance

(A

_cant

Test)

were

ele-ments.

RESULTS

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

lion/ml) of

(PEM)rats is depicted

80

60

C

TCT

N

CTCT

PEùI

lsr

tr

2nd

Effects of PEM on _{Sperm Quality and Spermatogenesis}

[image:3.595.176.539.66.826.2]

crcT

cTcc

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.}

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.

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.

Figure L

[image:4.595.249.545.53.699.2] [image:4.595.320.556.366.694.2]

Figure 4.

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

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.

CTCT

CTCT

N

PEI\,I

Figure 5.

C ₌ untreated control; TC -- placebo/treated control;

T ₌ treated, injectedwithTE.

Seminiferous tubule diameter

Mean and SD of seminiferous tubule diameter (pm) of

normal and protein energy malnutrition (PEM) rats

injected with TE, is depicted in Figure 6'

Med J Indones

250

C

TC

T

C

TCT

Figure 6.

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

A-Spermatogonium

Mean and SD of A-spermatogonium number/tubule of

normal and protein energy malnutrition (PEM) rats

injected with TE, is depicted in Figure 7.

C TCT C

TCT

N

PEM

Figure 7.

C = untreated conlrol; TC

-

placebo/treated control; T ₌ treated, injected with TE.

Pachytene spermatocYte

Mean and SD

of

pachytene spermatocyte number/

tubule

of

normal and protein energy malnutrition (PEM) rats injected with TE is depicted in Figure 8' Eliza et al.

1200

1000

800

600

400

200

2W

150

100

50

I

Vol 6, No 1, January - March 1997 _{Effects of PEM on}

Sperm _euatity and Spermatogenesis

C TCT C TCT

N

-E-'

the sperm _{concentration (p > 0.05) between TE in_} jected _{PEM and TE injected normal animals.}

Sperm viability

ropped al con-as well

Testis weight

ANOVA tesr for _{testis weight showed}

that

_{both in}

Testis diameter

S eminiferous tubule _diamete

r

[image:5.595.61.293.70.351.2]

A-spermatogonium Figure 8.

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

The

Statistical Analysis

Distribution _{and Homogeinity of data}

Before analysis of variance (ANOVA) test was carried

Sperm concentration

VoI6, No I, tanuary - March 1997 _{H uman R ec omb inan t Ery throp oi}

e tin 43 't6

14

12

10

I

6

4

2

0

2468101214

[image:6.595.57.557.61.749.2]

Weeks

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

Ht 30

25

20

15

10

5

o

Hb

12

10

I

6

4

2

0

14

12

10

I

l;Hbl

.

l*t,

I 4

2

0 Ht

2468101214

₁₆

Weeks

Figure 5. Case no. 5, male, 6g _{years old, Multiple Myeloma}

a a

4

6

''= I

10

12

Weeks

Figure 6. Mean Hb, Ht,from 5 cases

Hb

40

30

20

P achyt ene s P e rmut o cY t e

ANOVA

test

showed

that

both in normal (N) and

p-Èin

_"n"tgy malnutrition (PEM) animals TE injec-iion declineà- the number of pachytene spermatocyte

Gii'*'rry

G < 0.01) compaied to tn"

*ti,""ri$J.]

in

TE

in-al animin-als.

DISCUSSION

The declining of sperm concentration after TE injec-tion either on the normal (N) and protein energy

mal-"",iiii".

(PEM) animals is very likely' This is due to

"

t"g",it"

t"edÉack of exogenous testosterone directly io-inïrtvpophysis which

*pt"tt"t

the production of

irr

unaîsÉ

or may be through the hypothalamus so ,n"

ftoat",ion

of

dH-lH

decieases and eventually the

f.oâo"ti*

of

LH

and FSH

will

also drop'

In

both

cases,

finally

the process of spermatogenesis

will

be suppressed' Since there is no difference of sperm

con-centration between normal

(N)

and protein energy malnutrition (PEM) animals, there is no interaction

b",*""n

TE injection and protein energy-malnutrition

on spermatogènesis, so the declining

of

sperm

con-cent;tion

in this experiment is solely due to the TE injection.

The same explanation is hold true also for sperm mor-fnorogy, ,o

ih"

decreasing number-of. normal sperm

-orpnôtogy

was

due

to

the

TE

injection

that

hampered

IÉ" pro""tt of

organel cell .development'

Since there was no difference the number of normal

,f".-

*otpttology between TE injected in both normal

(fr)

ana pfur4 animats, so

it

was very likely that the à"ér"uring number of sperm morphology was due to the TE injection.

Thç

same results and very likely also the mechanism were also found on the viability of the sperm in the

sense that the number of viable sperm also declined

after TE injection in both normal and PEM animals;

this declining was also due to exogenous testosterone'

because there was no tnteraction between TE injection and PEM.

The most interesting phenomenon is that the viability

of

sperm in the untiéated control PEM animals also

declined,

although

in

the treated control

of

PEM

animals did

not.

This is probably due to the calory

deficiency; this deficience might be disrupt the cell

membrane with the end result affected the viability' In

the treated control of PEM, this deficience has been

Med J Indones

than in normal (N) grouP.

VoI6, No 1, January - March 1997

The histological preparations _of seminiferous tubules

taken from normal

(N)

and pEM animals show the

Effects of PEM on _{Sperm Quality and Spermatogenesis}

difference

of

the

rubules diameter (Fig. 9 and Fig.

10).

Figure 9' Histological preparation showing the seminiferous tubule of normal diet (N) animal; K (C), untreated control; Kp (CT),

treated,/placebo control; T _{(P), injected by TE. Compare the smaller tubule diameter of treated animal.}

[image:8.595.29.513.44.399.2] [image:8.595.19.529.67.684.2]

tn tr{n I CIO

Figure I0' Histological preparation showing the seminiferous rubule of protein energy malnutrition (pEM) animal; K (C),

t0 Eliza et aL

CONCLUSION

The effect of protein energy malnutrition (PEM) on spenn quality and spermatogenesis

of male rats

in-jected

with

TE, can be concluded as follow:

l.

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.

2.

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.

REFERENCES

l. Moeloek N. Kontrasepsi pria masa kini dan masa akan

datang. Presented at The VI Yearly Scientific Meeting,

In-donesian Andrological Society, Bandung, 1987.

2. Tadjudin MK. Cara keluarga berencana hormonal pada pria.

Proceeding

of National

Kongres

I,

Indonesian

En-d ocri nologi cal S oci ety, Jakarta, I 9 86;22-229.

3. Frick J, Danner Ch, Kunit G et al. Spermatogenesis in men treated with injection of mendroxyprogesterone acetate com-bined with testosterone enanthate. Int J Androl

1982;246-52.

4. Moeloek

N.

Penurunan kesuburan pria pada penyuntikan

testosteron (TE) + DMPA dan l9 Nortestosteron heksilok-sifenil propionat ( I 9-NT) + DMPA. Dissertation, Faculty of

Medicine University of Indonesia, Jakarta, 1991.

Med J Indones

5. Word Health Organization. Contraceptive efficasy

of

tes-tosterone induced azoospermia

in

normal men. Lancet,

1990; 336: 955-9.

6. Waites GMH. The research strategy of the WHO task force

on methods for the regulation of male fertility. In:

Perspec-tives in Andrology (Serio, M, ed), Raven Press, New York. 989;3-516.

7. Hamilton GD, Bronson FH. Food restriction and reproduc-tive development male and female mice and male rats.Am J

Physiol 1986; 250: 37 0-6.

8. Vawda AL, Mandlevana JG. The effect of dietary protein

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 Branch, Medan, Sumatra, 1965.

I 1. Sediaoetama AD. Faktor gizi. Bharata Karya Aksara,

Jakar-ta,1985.

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 treat-ment. Bio Rep 1991; 44:864-70.

15. Street C, Herry RJS, Al-Othman S, Chard T. Inhibition of

binding of gonadal steroid to serum binding protein by

P achyt en e s P ermat o cY t e

DISCUSSION

injection.

the TE injection.

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

Med J Indones

lvent (wijen oil). Acord-il contained 437o of

WA

essential fattY acid which mbrane integritY'

Our finding showed that both in normal (N) and PEM'

fe

ini""tià'n caused decrease of weight and diameter

oitt

"

t"ttit

and also seminiferous tubule diameter' As has been mentioned earlier,

TE

injection can sup-Dressed

the

spermatogenesis

through

feedback

It""ft*it*t

acËordingly the number of germinal cell

*irttin

the

tubules

*itt

utto

decreased' So

it

is

r"uto*tf"

that the diameter

of

the tubules and the àiurn"t", of the testis and also the weight of the testis

à""i"ut"a. But for PEM group another factor' protein ""r-r1y

^tl"utrion

(PEMI should be considered'

since therJ was a difference

between{E,iliiïîï"iffiï,:

grouP),

for all

three s diameter and tubule tatistical analYsis suPPort such preposition, since there was positive interaction betwËenlacbr TE injection and PEM'

According to T ermite and Tergui 14 reported that nutri-tional status seem to be an additional factor regulating

ir*-tt"roU

binding protein (SBP) level which may alter the percentage

of

SBP available for positive or

i"g",i*

ieedback; the decline of SBP' results in the inJreasine concentration

of

free androgen

in

serum' 'Stree*

eiat,l5

found that nonesterified

fatty

acids modify binding affinities

of

sex steroid hormone to SBP

t;

vitro' Since in this experiment PEM group is undernourish, it

is

predicted that supply of fatty acid

and

protein

is

very limited;

it

follows

by

the

avaitaUitity

of

SBP

in

the blood, more over

if

the affinity to testosterone

is

also

low,

then the blood free téstosterone increases sharply upon the introduc-tion of exogenous testosterone; followed by stronger feedback michanism in PEM group compared to

nor-mal

(N)

group. Thus,

this

condition

will

induce

,t

ong"i

ùppàssion

of

spermatogenesis

in

PEM than in normal (N) grouP.

The same explanation is hold true also for the number of A-spermatogonim and pachytene spermatocyte' so the decreasing number of both the germinal cells were 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 concerning to the number of germinal cells' it was very

I ikely thaithe decreasin g number of A-sperm atogoni a

and _iachytene spermatotyte in PEM animals was due