ELC TE MCC

SPERM FORMATION IN ZINC-DEFICIENT RATS

Marie-Claire ORGEBIN-CRIST M. FREEMAN G. H. BARNEY

Department of ^{Obstetyics and}

Gynecodogy

Vande y

bilt University ^School of ^Medicine Nashville, ^{Tennessee 33203}

SUMMARY

A quantitative histological study ^{of the} early changes ⁱⁿ the testes of 8 weanling ^{rats fed a}

zinc-deficient diet (0.5p.p.m. Zinc) ^{was made.} Eight ^control rats, pair-fed ^{to the deficient} rats, received 30 p.p.m. of Zinc. The earliest statistically significant change is the diminution of elon-

gated stage 3 spermatids (Roosen

Runge’s

classification) after 23 days ^of zinc deficient diet.

Since the number of stage ^{6 round} spermatids ^{after i6} days ^{of diet was not} statistically ^different

from the pair-fed control, ^it indicates that the earliest effect of zinc deficiency ^{on the} testis, ^is

an inhibition of the transformation of round spermatids ^into elongated ^{ones. These results are}

discussed in relation to the possible ^{role of zinc on} spermatogenesis.

INTRODUCTION

Since the work of

F OLLI S,

^{DAY and}

M C C OLLUM ( 1941 )

^and

^{M ILLAR , FisCHE R ,}

E L C OA T

E

and MAWSON

( 195 8, ig6o)

^it is known that adult male rats fed a zinc-defi- cient diet for 7 ^to 8 weeks

display

^various

pathological changes :

^marked

growth retardation,

^{loss of}

hair, alopecea

of the backs and

shoulders,

^{skin lesions and mar-} ked

parakeratosis

^{of the}

esophagus.

^In addition both the

spermatogenic

^{and endo-}

crine testicular function are

severely impaired

and the zinc concentration in the testes is reduced. All

changes except

the testicular

atrophy

^{are reversed}

by repla- cing

zinc in the diet.

According

^{to these}

authors, changes

^{in the} testicular endocrine function is not a

specific

consequence of the

zinc-deficiency

^{but is caused}

^by

^{a redu-}

ced food intake

resulting

ⁱⁿ inanition whereas testicular

atrophy

^is

specifically

^due

to an

inadequate supply

of zinc. The

present report

describes the

early histologic changes

^{in the testes} of zinc-deficient

weanling

rats, in order to elucidate the

genesis

of the testicular lesion.

METHODS

The 16 animals used in this study ^were part ^{of a} group of 46 rats used to study ^the genesis

of esophageal parakeratosis. ^{The result of this} part ^{of the} study ^{has been} reported separately (BARNEY, ORGEBIN-CRIST, MACAPINLAC, 1968).

Sixteen 21-day ^{old male} rats, ^{of the} Sprague-Dawley strain that averaged 44.8 g in weight

were divided in 2 groups. The first group of 8 ^{rats was} fed a. zinc-deficient diet (0-5p.p.m. zinc) containing in grams per ¹⁰⁰ g : zinc-low casein, ¹²⁰ (4) ; cottonseed oil, 2IO vitamin-sucrose

mix, 3 5

; mineral mix !3.55 ; ^choline chloride, o-15 ; and sucrose, 561.3 ; ^{a second} pair-fed group of

8 animals was offered the same diet supplemented ^with ZnS04. ! H20 ^to provide ^{an additional}

30

p.p.m. of zinc. All animals ^were housed in plastic cages designed ^{and made} locally$. ^{Diet was}

fed in plastic cups and deionized water ^was provided ad libitum from plastic ^bottles. Starting

on the sixth day, ^the daily food intake of the pair-fed group ^was adjusted ^to equal ^{the mean}

intakes of the animals fed the zinc-deficient diet. One testis from 2 zinc-deficient animals and

2 pair-fed controls killed on days 9, 16 and 23 of the experiment ^{was examined} histologically,

and the other testis was removed for zinc analysis. ^On day 30, ² zinc-deficient rat were repleted with I mg of zinc (ZnS04.7 7 H20) by intraperitoneal injection ^after hemicastration. They ^were

fed the control diet for 14days before removal of the other testis One of the zinc-deficient rats died and results are not included.

The esophagi ^for light ^and phase microscopy ^{were fixed in 10}p. ¹⁰⁰neutral formalin. Testes

were fixed in Bouin’s fluid, embedded in Paraplast ^{and cut into} 7Vsections. Zinc was determined in testes by ^atomic absorption spectroscopy. ^{Testes were} prepared ^for analyses by ^a wet-ashing procedure

7 .

In each animal the early primary spermatocytes, ^{the old} primary spermatocyte ^{and the} elongated spermatids ^were enumerated in 120 cross sections of seminiferous tubules showing

the cellular association of stage 3

(Roosen-Runge’s

classification, ^{ROOSEN-RUNGEand} GIESEL, 1950

). ^{The Sertoli} cells with the nucleolus in the plane ^{of the} section, the round and the elongated spermatids ^were enumerated in 60cross sections of tubules in stage 6. The counts were averaged per animal and per group and expressed ^as the number of cells per tubular cross section. To take into account the difference in nuclear diameter Abercrombie’s formula was applied. (ABERCROMBIE,

1946).

TC ⁼ true count, ^{CC = crude} count, ^{S = section} thickness and d ⁼ nuclear diameter.

In order to adjust the germ cell count for shrinkage ^{of the} seminiferous table the Sertoli cell correction factor proposed by ^{CLERMONT and} MORGfiNTHALER, (1955) ^was used when the number of Sertoli cells with the nucleolus in the plane of the section was significantly different between the control and zinc-deficient _group.

RESULTS

Body weights

and testicular zinc concentration at the time of sacrifice are indi- cated in table i. Zinc concentrations in the testis are

variable,

but the average ^concen- tration in the zinc-deficient _group is

statistically

^{different from the}

pair-fed

^control.

( 1

) ’Case in purified ; Nutritional Biochemicals Corporation, Cleveland. 2Vitamin A, ^{I ooo IU} (Aquasol A, U. S. Vitamin and Pharmaceutical Corporation, ^New York), ^vitamin DQ, 125 IU (Drisdol, Winthrop Laboratories, New York), ^and a-tocopherol, ⁶⁰ mg (Nutritional Biochemicals Corporation) ^{were added to}

10g cottonseed oil (Wesson Oil, Hunt-Wesson Foods, Fullerton, California). ’Vitamin-sucrose Mix, mg/kg

of mix : thiamine.HCI, zoo; riboflavin. 120 pyridoxine.HCI, 80; ^Ca pantothenate, 320; biotin, 4; 3 nicotinic acid, 300 ; folic acid, Io ; vitamin B12, 0.40 : menadione, 6.6 ; ^{and sucrose to make i ooo} g.’Each

3.55 g of mineral mix contained : (g) CaHP04, 2.58 ; KCI, 0.3433 NazC03> 0.115 ; MgS04· ⁷ H20, o.4og ; §

FeS0 4’

7 H.0, 0.06 ; MnS04·H80, 0.031 ; CoCi2.6 H20, 0.004; CuSO,-6H!O, 0.006 ; ^K I, ^o.oo0 4; § and NaF, o.oo0 8. ’Commercial cane suggar. Godchauz Sugar Refining Company, ^New Orleans, ^Louisiana.

6

Economy Plastics, Nashville, Tennessee. ’Analytical ^Methods for ^Atomic Absorption Spectrophotometer Manual 990-9461, 1964 Perkin-Elmer Corporation, Norwall, Connecticut.

Annales de Biologie ^{animale. -} ig7i.

Parakeratosis

(one

of the earliest

symptoms

^of

zinc-deficiency

^{MILLAR et al.}

( 195 8)

was evident in one of the rats after 16

days

of diet and in all the rats thereafter.

After zinc

repletion

^the

esophagus

^{was normal.}

After nine

days

of diet the rats were 30

days

old. Round

spermatids

^{but no}

elongated spermatids

^were

present

in the control testes. In the testes of the zinc- deficient rats the number of germ cells ^{was not}

significantly

different between the control and the zinc-deficient group, the difference in the number of

pachytene spermatocyte being

^at the limit of the

significance (t

⁼

0 . 05 ) (table 2 ).

After 16

days

of zinc-deficient diet there was no

significant

difference in germ cells counts between the control and deficient _group, the difference in _young

pri-

mary

spermatocyte

^number

being

^at the limit of the

significance (t

⁼

0 . 05 )

(table 3 ).

^In both control and zinc-deficient testes

elongated spermatids

^had

appeared.

^{One of the} zinc-deficient rats showed

signs

^of

parakeratosis

^{in the eso-}

phagus indicating

^{that the}

zinc deficiency

^had

already

^taken

place.

After ₂₃

days

of zinc-deficient diet both rats showed

signs

^of

esophageal

para- keratosis. The overall

picture

^{of the testes in} both groups ^was similar. A few

dege-

nerate tubules were seen but

they

^{were in the same}

proportion

in each group. The number _{of young}

primary spermatocytes

^{and round}

spermatids (stage 6)

^{were not}

different between the control and

experimental

group. The number of

pachytene

spermatocytes

^and

elongated spermatids (t

⁼

0 . 001 )

^was

significantly

^different

(table 4 ).

^{This was confirmed}

by

^the presence of _very few _sperm in the lumen of the

epididymal

canal of the zinc-deficient rats in contrast to the _many sperm in the

epididymides

^{of the}

pair-fed

^control.

After 30

days

^of diet the difference in the numbers of young

primary

sperma-

tocyte

^and

elongated spermatids

^{between the} control and

experimental

group ^was

highly significant (t

⁼

o.ooi.)

^{There was no} difference in the number of

pachytene spermatocytes

and round

spermatids (table 5 ).

^{There were a} number of tubules which showed a

non-specific type

^of

degeneration

^with

pyknotic spheres,

^exfolia-

tion of

cells,

^{and the formation of} multi-nucleated

spermatids. Only

^{tubules not}

showing

^this

non-specific type

^of

degenerescence

^{were counted. In the}

epididymis

of _{30 and 3i}

-day

^{old rat, no}

spermatozoa

^are

present

in the lumen of the

epididymal

canal. In the

epididymis

^of the 4q. and

51 -day

^old zinc-deficient rat

(on day

23 and

30 )

very few sperm could be ^seen

compared

with those of the

pair-fed

^controls

which were

packed

^{with sperm.}

DISCUSSION

In view of the results

reported ^above,

^{the effect of zinc}

deficiency

ⁱⁿ the various

phases

^{of the formation of the}

spermatozoa

will be discussed.

This

will be facilitated

by

^the

knowledge

of the duration of the seminiferous

epithelium cycle

^{and its}

^diffe-

rent

stages

from the work of

CI,!RMONT, I,!BI,OND

and M!ISSI!R

(i 959 ),

^{It is}

possible

to follow the

progression

^of germ cells at

different

time-intervals : for

example,

^the

leptotene spermatocytes

ⁱⁿ

stage

3

counted

^on

day

9 of the

experiment

^will

give

^rise

to

pachytene spermatocytes

ⁱⁿ

stage

7 ^on

day 1 6,

^{to round}

spermatids

ⁱⁿ

stage

^{5 on}

day

23 and to round

spermatids

ⁱⁿ

stage

^{8 on}

day

^30.

S f

iermalogonial

^divisions

In

only

^{2 of 8} zinc-deficient rats the number of _young

primary spermatocytes

was reduced. On

day 1 6,

the difference ^was at the limit of the statistical

significance (t

⁼

0 . 05 )

^{and the}

daughter

cells of these

preleptotene spermatocytes (pachytene spermatocytes

^at

day

23 and round

spermatids

^at

day 30 )

^{were in same numbers}

in

pair-fed

control and zinc deficient rat

casting

further doubt on the

significance

^of

the difference observed at

day

^16.

In conclusion

during

the first 30

days

of zinc deficient diet and with one pos- sible

exception (one

^{rat at}

day 30 )

the number of

preleptotene spermatocytes

^are alike in the control and Zn deficient _group and one can assume that the

spermato- gonial

^{divisions were not affected}

by

^the zinc-deficient treatment.

Meiotic

pro_phase

In

only

^{one of 8} zinc-deficient rats the number of

pachytene spermatocytes

was reduced. This was on

day

23, but the

daughter

cells of these

pachytene

sper-

matocytes (round spermatids

^at

day 30 )

^{were in same} numbers in the control and zinc-deficient group. The meiotic

prophase

^{does not seem to} be affected

by

^the

zinc

deficiency

^treatment

during

^{the first} 30

days

^{of the treatment.}

Reduction divisions

There was no difference in the numbers of round

spermatids

in all the zinc deficient rats

indicating

that the reduction divisions had taken

place normally.

Spermatid elongation

At

day

9 of the

experiment

^no

elongated spermatids

^were

present.

^At

day

¹⁶ their number was not

significantly

different between the control and the

experimental

group : the

symptoms

^{of zinc}

deficiency

^were

just developing. Thereafter, although

the number of round

spermatids

^{was not}

different,

^{all the counts of}

elongated

sper- matids were

significantly

different in all the zinc-deficient rats as

compared

^{to the}

pair-fed

^control

rats, demonstrating

^{a severe} inhibition of the transformation of the round

spermatid

^{into the}

elongated,

^one very

early

^{in zinc}

deficiency

^{and in all}

the rats studied.

Zinc,

^a characteristic constituent of the male

reproductive

organs and of semen,

plays

^an

important

^{but not}

fully

understood role in

reproduction (M ANN , 19 6 4 ).

^In

plants

^and

microorganisms

zinc has been shown to be involved in nucleic acid and

protein metabolism,

the earliest biochemical lesion in zinc

deficiency being

^a

depres-

sion in RNA

synthesis,

^followed

by

^{a reduction in}

production

^of

protein

^{and DNA}

(WINDER

^and

D!rrrnErrY, 1959 ;

^{SCHNEIDER and}

PRICE, i962 ; ^WACg!R, i 9 62 ;

^§

WINDER and

O’Ii AR a, 19 6 2 ;

^{WEGNER and}

ROM ANO , rg6 3 ).

^{In testes} of zinc-deficient rats

M A C APINLA C, PE AR S ON ,

BARNEY and

DARBY, ( 19 68),

^{SOMERS and UNDER-}

WOOD,

( 19 6 9 ),

^{were unable to detect}

impairment

^{of RNA}

synthesis

^{but found an} increase in

protein

^{and RNA} catabolism in zinc-deficient testes.

During spermato-

genesis

^{RNA is}

progressively

lost from the

developing

germ cell

(C ASP E RSSON ,

Ig39 ! 1

BRACKET, igq. 7 ).

^RNA

synthesis stops

^{soon after} the second meiotic division in very

early spermiogenesis (M ONESI , i 9 6 5 ).

Therefore the most

likely

^{cells to be}

inju-

red

during

^zinc

deficiency

^{would be}

spermatogonia

^and

spermatocytes

^{where both} RNA content and RNA

synthesis

^is

higher

than in the

spermatids. Although

^this

may very well occur later in zinc

deficiency,

the first germ cells affected in ^our expe- riment were the

spermatids

^{and more}

specifically

^the

spermatids undergoing

^the

process of

elongation.

In this

regard,

zinc has been shown to be

present

ⁱⁿ

high

concentrations in sperm

(MA NN , r 945 ;

^{MAWSON and}

FISHER, i 953 ). WETTE RD A L E, ( 195 8)

showed that the testis of rats attained maximum 65Zn level about ⁱ week

following isotope injec-

tion. Maximum concentration of e6Zn in both

caput

^{and cauda}

epididymidis appeared approximately

^{2 and} 3 weeks

respectively

^after

injection

^of

&dquo;Zn, suggesting

^that

the element was

incorporated

^{in late}

spermatids

^and

transported

^{from the testis}

into the

epididymis by

^the

spermatozoa.

These observations have been confirmed

by

^others

(WAi!r,!y, Mol!>,ATT,

^{CROOK and}

MALLARD, i 9 6o ; M ILL A R ,

^VINCENT and

MA W SO N , I9 6 I ; G UNN , G OULD

^and

A ND E R SO N , 19 63).

^More

recently PAR IZ EK, B

OURSNELL

, H AY ,

^{BABICKY and}

T A y r, OR , ( 19 66), studying

^{the zinc content of the}

maturing

^rat testis showed that

during

the first month of life the zinc concentration remained

fairly

^constant.

During

the second month of life the concentration of testi- cular zinc increased

considerably.

^This increase coincides

roughly

^{with the time}

when

spermatids

^are transformed into

spermatozoa,

^{and the authors}

postulated

that this increase could well be related to the formation of intracellular zinc-metallo

proteins including

^certain

zinc-containing

enzymes in

spermatozoa.

Indeed we would conclude from the

present study

that zinc is _necessary

during

this critical

period

^{since rats fed a} zinc-deficient diet exhibit normal

spermatogenesis

up ^to the

elongation

^of

spermatids. However,

^{the final}

phase

^of

spermatogenesis

is inhibited in the absence of zinc.

Reçu pour publication ^{en mai 1971.}

ACKNOWLEDGMENTS

The authors would like to thank Dr. A. MEANS for reading ^this manuscript. ^{The work was} supported by ^Grant HD-oo673 and Career Award Ky-HD-38, ⁸⁸² from the National Institute of Health and Human Development, ^the Population Council and the Ford Foundation.

RÉSUMÉ

SPERMATOGENÈSE

CHEz DES RATS

CARENCES

EN ZINC

Une étude histologique quantitative ^des modifications précoces ^{dans les} testicules de 8 Rats de

21 jours ^{soumis à un} régime ^{carencé en zinc} (0,5 p.p.m. de zinc) ^{a été} menée. 8 Rats témoins

reçoivent 30 p.p.m. de zinc. La modifica.tion la plus précoce, statistiquement significative ^{est la}

diminution des spermatides allongées ^du stade 3 (classification ^de

Roosen-Runge)

après 23jours

du

régime

^{carencé en zinc.}

Comme le nombre de spermatides rondes du stade 6 après ⁱ⁶ jours ^de régime ^n’est pas sta-

tistiquement ^différent par rapport ^{aux animaux} témoins, ^il apparaît que le premier ^{effet d’une}

carence en zinc sur le testicule est l’inhibition de la transformation des spermatides ^{rondes en} spermatides allongées. ^{Les résultats sont discutés en fonction d’un rôle} possible ^{du zinc sur}

la spermatogenèse.

REFERENCES

F OLLIS

, ^R. H., ^{DAY H.} G., McCoLLUas E. V., 1941. Histological studies of the tissues of rats fed a

diet extremely ^{low in zinc.} J. Nutri., 22, 223. M

ILLAR M. J., ^{FISCHER M.} I., ^{ELCOATE P.} V., ^{MAwsoN C.} A., 1958. The effects of dietary ^{zinc defi-} ciency on ^the reproductive system ^{of male rats. Can.} J. ^Biochem. Physiol., 36, 557.

M

ILLAR M. J., ^{ELCOATE P.} V., ^{FISCHER M.} I., ^{MAwsoN C.} A., 1960. Effect of testosterone and gonadotrophin injections ^{on the sex} organ development of zinc-deficient male rats. Can. J. ^Biochem.

Physiol., 38, 1457.

B

ARNEY G. H., ^{ORGEBIN-CRIST M.} C., ^MACAPINLAC M., 1968. Genesis of esophageal parakeratosis

and histologic changes ^{in the testes} of the zinc-deficient rat and their reversal by ^zinc repletion. J.

Nutri., 95, 526.

RooSEN-RuNGE E. C., ^{GIESEL L.} 0., 1950. Quantitative ^{studies on} spermatogenesis in the albino rat. Am. J. Anat., 87, ^1.

A

BERCROMBIE M., 1946. Estimation of nuclear population from microtome sections . Anat. Rec., 94,

23 8.

CL

ERMONT Y., ^MORGENTALER H., 1955. Quantitative study of spermatogenesis ^{in the} hypophysec-

tomized rat. Endocrinology, 57, 369. C

LERMONT Y., ^{LEBLOND C.} P., ^MEissiER B., 1959. Duree du cycle ^de 1’epithelium seminal du rat.

Arch. Anat. Morph. Exper., 48, 37.

M

ANN T., 1964. ^The biochemistry of ^{semen and} of ^{the male} reproductive ^tract. Methuen, London.

WINDER E., ^DENNENY J. M., 1959. Effect of iron and zinc on nucleic acid and protein synthesis ⁱⁿ Mycobactérium smegmatis. Nature, Lond., 184, 742.

S

CHNEIDER E., ^{PRICE C.} A., Ig62. Decreased ribonucleic acid levels : a possible ^{cause of} growth ^inhi-

bition in zinc deficiency. ^Biochem. Biophys. Acta, 55, 406.

W

ACKER W. E. C., 1962. Nucleic acids and metals. III. Changes in nucleic acid, protein, ^{and metal}

content as a consequence of zinc deficiency ⁱⁿ Euglena gracilis. Biochemistry, 1, 859.

WINDER F. G., ^O’HARA C., 1962. Effects of iron deficiency ^{and zinc} deficiency ^{on the} composition of Mycobactérium smegmatis. Biochem. J., 82, 98.

W

EGENER W. S., ^{ROMANO A.} H., 1963. Zinc stimulation of RNA and protein synthesis ⁱⁿ Rhizopus nigricans. Science, 142, 1670.

WEG E N

ER W. S., ^{ROMANO A.} H., 1964. Control of isocitratase formation in Rhisopus n%gricans. J.

Bacteriol., 87, 156.

M

ACAPINLAC M. P., ^{PEARSON W.} N., ^{BARNEY G.} H., ^{DARBY W.} J., 1968. Protein and nucleic acid metabolism in the testes of zinc-deficient rats. J. Nutr., 95, 569.

SO M

ERS M., UNDERWOOD E. J., 1969. Ribonuclease activity and nucleic acid and protein meta- bolism in the testes of zinc-deficient rats. AusE. J. Biol. SCi., 22, 1277.

C A S

PERSSON T., 1939. Uber Die Rolle Der Desoxyribosenukleinsgure ^{Bei Der} Zellteilung. ^Chromo-

soma, 1, 147.

B

RACHET J., 1947. Nucleic Acids in the Cell and the Embryo. Symp. ^Soc. Exp. Biol.,1, 207. Cambridge University ^Press.

M

ONEST V., 1965. Synthetic activities during spermatogenesis ^{in the mouse.} Exp. Cell. Res., 39, 19

7.

M

ANN T., Igq.g. Studies ^on the metabolism of semen. I. General aspects occurrence and distribution of cytochrome, certain enzymes and coenzymes. Biochem. J., 39, 451.

M A

wsoN C. A., FISHER M. I. 1953. Zinc and carbonic anhydrase ^{in semen.} Biochem. J., 55, 6

9 6.

W

ETTERDALE B., 1958. Experimental studies on radioactive zinc in the male reproductive organs of the rat. Acta Radiol., Suppl., 156, ^I.

WAK E L

EY J. ^C. N., ^MOFFATT B., ^CROOK A., ^MALLARD J. R., 1960. The distribution and radiation

dosimetry ^of Zn-65^{in the} rat. Int. J. Appl. Radiat., 7, 225.

M ILLAR

, ^M. J., ^{VINCENT N.} R., MAWSON C. A., 1961. ^An Auto radiographic study ^of the distri- bution of zinc-65 in rat tissue. J. Histochem. Cytochem., 9, ^111.

G

VNN S. A., ^GOULD T. C., ANDERSON W. A. D.,

1 9 6 3 .

Distribution and movement of zinc in the rat testes. Arch. PaEho6., 75, zi.

P ARIZ8

K J., ^BOVRSNELL J. C., ^{MAY M.} F., BAB1CKY A., ^{TAYLOR D.} M., 1966. Zinc in the maturing

rat testis. J. Reprod. Fert., 13, 5or.