Mutation Research, 3° (1975) 267-272 © Elsevier Scientific Publishing Company, A m s t e r d a m - - P r i n t e d in The Netherlands

. 267

ABSENCE OF ACROSOME: AN E F F I C I E N T TOOL IN MAMMALIAN MUTATION R E S E A R C H *

J. MOUTSCHEN AND A. COLIZZI

Laboratoire de Gdndtique, Umvers,td de L#ge, ~5, rue Forguer, B-4ooo Linage (Belgique) (Received January I4th, 1975) (Revision received May 7th, 1975) (Accepted May i7th, 1975)

SUMMARY

Spermatozoa without acrosomes ("acrosomeless") have been qualitatively and quantitatively analysed in several strains of mouse. The frequency of these abnormal cells was fairly constant within the same strain, but increased with age, consanguinity and after treatment of mice at pre-meiotic stages with alkylating agents. From this behaviour, it is thought that these abnormal cells are true mutants which could be used in mutation research.

INTRODUCTION

The heritability of sperm characteristics is an established fact. BEATTY2 proved that some gross anomalies are due to autosomal mutations whereas others could be related with the action of the Y chromosomelL It is also clear that some characteristics are under polygenic control. The advantages of sperm defects, especially acrosome abnormalities are: (i) such defects could be efficiently recorded on a large scale and (2) the analysis could be carried out routinely and inexpensively in various mammalian species. The genetics of size and shape of spermatozoon organelles has been surveyed ~. The variation of sperm between species is large, but tends to be within a narrow range in the same species. In principle, defects of each organelle: head, acrosome, middle piece and main tail piece could be exploited for mutation work. Even enzymatic reactions might be used. In the mouse, the overall frequency of abnormal spermatozoa is high in some strains (up to 21% in C57) but low in others (6% in CBA)~, 9-1~. In a recent research on mice treated with alkylating agents we observed an unexpectedly high increase of acrosomal abnormalities which prompted us to investigate the possibility of using this organelle in mutation work. In the bull, an abnormality of acrosomes associated with head defects has been mentioned 13 and a specific acrosomic deformation described e. There is evidence for its genetic nature in the bull (recessive gene kn)L in * Recherches partiellement r~alis6es grace ~ un subside du Minist~re de la Sant~ pubhque de Belgique.

268

J . M O U T S C H E N A N D A. COLIZZI

which species most acrosome abnormalities seem to be associated with sterility~ as they are in the boar I. BRADENa also observed strain differences in the mouse and first gave evidence of genetic effects. Genetic variations of acrosomes have been described between CBA/Fa, V/Be, J B T / J d and C57 B L / F a (see ref. 3). The purpose of the present article is to investigate the possibility of using acrosome abnormalities in mutation research and to select specific defects which could be unambiguously identified and correlated with a genic action. MATERIAL AND METHODS

The following mouse strains were selected for the present investigation. I. Q strain (from FALCONER, Edinburgh), some lines of which served in the past for selection experiments. This strain was described in a previous paper'. II. Tailless mice T/te (from Harwell) and T/t12 (from Columbia University). I I I . c57 BL and C57 BR. For treatment with chemicals, mice were injected intraperitoneally (0.5 ml) with methyl methanesulphonate (MMS), ethyl methanesulphonate (EMS) and isopropyl methanesulphonate (iPMS) (Eastman Kodak) at the dose of 25 mg/kg. Mice were killed b y dislocation of the neck. Sperm were collected b y gently squeezing the ductus deferens into o.15 M NaC1 and smearing the suspension on a clean slide. Two or three slides were prepared per animal. After being dried, spermatozoa were fixed for i h in IO~o formalin and then stained b y a slightly modified PAS as follows: immersion in periodic acid (5%) for 5 min or more, washing with running distilled water, dipping for 15 min in Schiff reagent, then in three successive SOs water baths (2 rain each), rinsing in running distilled water, then mounting in Canada Balsam. A modified Giemsa technique 8 that gives a delicate staining of the acrosome area does not seem appropriate for the present purpose. Duration of treatments and concentrations described here are optimal for mouse, but should be slightly modified for other species. Cells were observed with a Zeiss photomicroscope having a green interference filter, under phase contrast or Normarski interference. Lengths and widths of acrosomes measured b y planimetry were taken as parameters b y BEATTY~. In the present investigation, a Zeiss Micro-videomat was coupled to the photomicroscope for quantitative measurements. Since the mouse acrosome is sickle shaped, acrosome area was preferred to length and width. Use of the Micro-videomat avoids the time-consuming planimetric method and is more accurate. We consider as acrosome only that part of the head which is positively stained b y PAS and thus easily discriminated with the Micro-videomat from the post-acrosomic area. This acrosomic area is expressed as a proportion of the total head area because the size of the head/zaries. This measurement could be performed as a routine test. Details will be given in the next section. For qualitative detection of acrosomeless cells the Micro-videomat is not required. RESULTS

It was much more difficult to analyse the mutant cells in the epididymis since the shape of the acrosome is very different. Therefore, the analysis was restricted to the ductus deferens.

QUANTITATIVE ANALYSIS OF SPERMATOZOA

269

All a b n o r m a l acrosomes o b s e r v e d in all mouse strains i n v e s t i g a t e d so far fell into four groups: (i) Acrosomes swollen, less stained, with a w a v y edge. (2) Acrosomes n o r m a l l y stained, b u t w i t h a notch visible at the rear edge (type p r e v i ously described3). (3) Acrosomes d i s t i n c t l y overhanging the apex. (4) Acrosomes c o m p l e t e l y a b s e n t with smaller h e a d (post-acrosomic area). These cells will be referred as "acrosomeless". W e observed t h a t the frequency of the t h r e e first t y p e s was v a r i a b l e a n d could o n l y be s u b m i t t e d to a t i m e requiring, q u a n t i t a t i v e analysis. F o r the sake of simplicity, t h e fourth t y p e only was t a k e n into account. The m u t a n t origin of these cells was n o t at first evident, a n d all possibilities of a r t i f a c t s should be eliminated. T h e smearing conditions were i n v e s t i g a t e d a n d found to be w i t h o u t influence on the f r e q u e n c y of such a b n o r m a l cells.

*

x

Fig. I. (I) Normal head with acrosome stained with PAS. (I') Abnormal head without acrosome. (2) Normal head area discriminated with Micro-videomat. (2') Abnormal head area (smaller) discriminated with Micro-videomat at the same level as the normal head of (2). (3) Discrimination of the acrosome area (indicated by the arrow) of a normal head. (3') Discrimination of the very small area (indicated by the arrow) of an acrosomeless head (at the same discrimination level as the normal acrosome of (3)). Enlargement of all spermatozoa 3200 ×.

Fig. I . I ' . gives an e x a m p l e of an acrosomeless s p e r m a t o z o i d versus a n o r m a l t y p e (Fig. I.I). I n t h e a b n o r m a l acrosomeless cells, the M i c r o - v i d e o m a t allows to discrim i n a t e a v e r y small a r e a (Figs. 1.2' a n d 1.3' ) as c o m p a r e d with the control (Figs. 1.2 a n d 1.3). The n a t u r e of this u n s t a i n e d a r e a is not y e t elucidated. Because it always remains u n s t a i n e d after PAS, it is u n l i k e l y to contain acrosomic components. D a t a from c o m p a r a t i v e m e a s u r e m e n t s at t h e critical discrimination level are given in Table I. In the n o r m a l series, v a r i a t i o n of size of the acrosomic area was large which suggests the existence of a polyallelic series of genes governing its size. The s t a t i s t i c a l difference between " n o r m a l " a n d acrosomeless is h i g h l y significant. Table I also shows t h a t the size of t h e h e a d is significantly smaller in m u t a n t cells. W h e n the acrosomic area is expressed as a percentage of the t o t a l h e a d area, t h e v a r i a b i l i t y decreases, b u t the difference r e m a i n s h i g h l y significant. The m e a n value of the h e a d area is of the same order of m a g n i t u d e as r e p o r t e d before a. I n contrast,

270 TABLE

j. MOUTSCHEN AND A. COLIZZI I

M E A N A R E A (]~2) O F A C R O S O M E S

Normal Acrosomeless

IN NORMAL

AND ABNORMAL

CELLS

Mean acrosome

t o.o 5 a

Mean head

t o.o5 a

Proportions (%)

2.46 0.26

~ o.128 :J: 0.068

22 80 12.86

± I.O :[: o.18

lO.79 2.02

3° measures in each case in Q strain. TABLE II FREQUENC~ r OF ACROSOMELESS

Strain

CELLS IN VARIOUS

Q

Q (Tabby)

Number of acrosomeless ceils 72 Percentage 1.8 Confidence limitto.o5 a 0.6o

315 7.88 o.91

STRAINS

Tailless T/t6

T/t12

94 2.35 0.52

14 o.35 o.24

C57 B L

C57 B R

Mean

i 13 2.82 0.44

199 4.97 0.74

134.5 3.36

Each mean established on two slides (2 × 2000 cells) five-month-old males. t he m e a n v al u e of the acrosomic area is m u c h lower in the present data. This m i g h t be because we only m e a s u r e d the P A S - p o s i t i v e region in c o n t r a s t w i t h previous work s. In Tab l e II, th e f r e q u e n c y of acrosomeless cells was a n a l y s e d in several strains at an a r b r i t r a r i l y chosen age. T h e v a r i a t i o n of t h e f r eq u en cy b e t w e e n strains was r a t h e r large. No correlation was established b e t w een the f r e q u e n c y of a b n o r m a l cells a nd t h e overall fertility of th e strain. Males, carriers of t h e sex-linked T a b b y gene, always showed an u n e x p e c t e d high frequency, whereas males of t h e balanced lethal s y s t e m T/tl2 surprisingly h a d the lowest f r e q u e n c y so far recorded. In t h e latteI strain, inbreeding h a d been kept at t h e lowest possible level. Thus, inbreeding was t h o u g h t to p l a y a positive role and was therefore i n v e s t i g a t e d in t h e Q strain. Table I I I shows t h a t t h e higher the f r e q u e n c y of a b n o r m a l cells, the higher the inbreeding i ndex (sample correlation coefficient r = 0.946 ). These d a t a suggest t h a t b y s y s t e m a t i c out b reed i n g t h e f r e q u e n c y of the a b n o r m a l t y p e s could be minimized. TABLE III VARIABILITY

OF THE FREQUENCY

Ammal

i

Number of aerosomeless cells Percentage Inbreeding index, %

OF ACROSOMELESS

2

3

4

SPERMATOZOIDS

5

6

IN STRAIN

7

8

TABLE IV NUMBER

9

112 62 33 31 28 51 21 42 48 2.8 1.55 0.83 0.78 0-70 1.28 0.53 I.O5 1.45 20. 3 1.25 3 . 1 3 3.i~3 3.13 7.8 2.34 6.2 12. 5

4ooo cells analysed for each animal.

INCREASED

Q (4 months)

OF ACROSOMELESS

Age (months)

Number of mutant cells Percentage Confidence limit (t o.o5a )

3.5

6z i. 55 i 0.36

40oo cells analysed at each age.

CELLS WITH

5

72 I. 80 :[: o.61

AGE IN Q STRAIN

7.5

83 4.5 :t: 0.90

II

137 6.80 i i.ii

io

lO4 2.60 18. 7

Q U A N T I T A T I V E A N A L Y S I S OF S P E R M A T O Z O A

271

Another question that arose was whether, for the same strain and almost the same consanguinity level, the frequency of the aerosomeless cells increased with age. Table IV shows that this was so in the Q strain from 3-5 to I I months. It is not yet known whether a plateau is reached for older animals or whether the frequency of abnormal cells is still increased. On the other hand, the animals were never analysed before puberty, at which time a large variability of abnormal cells has b~en reported in the literature TM. TABLE

V

EFFECTS OF THREE ALKYLATING AGENTS ON THE FREQUENCY OF ACROSOMELESS CELLS, 31 DAYS AFTER INJECTION (20 m g / k g ) IN Q STRAIN

Control EMS MMS iPMS

Mean ( % )

Confidence hm~t t 0.05

1.O5 1.25 II.I 16. 7

:L -4~2 ~-

0.32 O.40 1.29 1.90

2ooo cells a n a l y s e d in e a c h case.

The suitability of the method was tested after mice had been injected with mutagens. Table V gives examples of the effects of alkylating agents on the frequency of acrosomeless cells counted 31 days after i.p. injection (treatment at pre-meiotic stages). At that dose no significant difference from the control was found for EMS whereas the frequencies of abnormal cells after MMS and iPMS were considerably enhanced. Other stages of spermatogenesis are under investigation in an attempt to explain the differential response towards the chemicals. DISCUSSION

The main problem arising from the present investigation is the nature of acrosomeless spermatozoids. Although a variety of mechanisms can give rise to abnormal cells, there are several reasons for the belief that they are "true mutants". First, there was a constant frequency of these cells within the same ductus deferens, whatever the smearing conditions. Secondly, the frequency within the same strain and its enhancement with consanguinity and age was constant. Lastly, there was a highly significant increase after injection of chemicals known to be powerful mutagens. However, it is not yet known whether genes determining acrosome characteristics can act in the haploid post-segregational stages. The high frequency of acrosomeless cells in some strains m a y possibly be related to a polygenic origin of the acrosomic characteristics. In this respect, no simple segregation of the absence of acrosome was observed. One would not expect such acrosomeless spermatozoa to be functional (as stated for bull and boar 1) although this point can hardly be experimentally verified.) The exceptionally high frequency of abnormal cells recorded in T a b b y males is not the consequence of a high inbreeding index but indicates a possible interaction of the T a b b y gene itself. Ultrastructural studies would be required to bring more knowledge about the nature of the cell abnormalities described above. However, in recent experiments we

272

J. MOUTSCHEN AND A. COLIZZI

c o n f i r m e d t h e s u i t a b i l i t y of t h e t e s t . A r e l a t i o n s h i p w a s e s t a b l i s h e d b e t w e e n t h e freq u e n c y of a c r o s o m e l e s s cells a n d t h e d o s e of e°Co 7 - r a y s ( 5 - 1 6 o r a d ) a f t e r i r r a d i a t i o n of s p e r m a t o g o n i a l cells ( u n p u b l i s h e d d a t a ) . The technique reported here could be extended to other acrosome anomalies and t o o t h e r m a m m a l s . O u r first o b s e r v a t i o n s s u g g e s t t h a t species s u c h as C h i n c h i l l a a n d g o l d e n h a m s t e r a r e f a v o u r a b l e m a t e r i a l s w h e r e a s o t h e r s s u c h as r a b b i t a n d g u i n e a - p i g a r e less s u i t a b l e . REFERENCES I BANE A., Acrosomal abnormality associated with sterility in boar, Proc. I V Int. Congr. Atom. Reprod., The Hague, (1961) 81o-817. 2 BEATT¥, R. A., The genetics of the mammalian gamete, Bwl. Rev., 45 (197o) 73-119. 3 BEATTY, R. A., The genetics of size and shape of spermatozoon organelles, Proc. Int. Syrup. The genetics of the Spermatozoon, (1972 ) 97-115. 4 BRADEN, A. ]7I. W., Strain differences in the morphology of the gametes of the mouse, Aust. J. Biol. Sc,., i2 (1959) 65-71. 5 BROZEK, C., Proportion of morphologically abnormal spermatozoa in two inbred strains of mice, their reciprocal F 1 and F 2 crosses and backcrosses, Acta Biol. Cracov (Ser. Zool.), 13 (197o) 189-198. 6 DONALD H. P. AND J. L. HANCOCK, Evidence of gene-controlled sterility in bull. J. agric. Sc~.. 43 (1953) 178-181. 7 FALCONER D. S., Rephcated selection for body weight in mice, Genet. Res. Camb., (1973) 291321. 8 HANCOCK J. L. AND D. J. TREVAN, The acrosome and post-nuclear cap of bull spermatozoa, J. Roy. mwr. Soc., 76 (1957) 77-83. 9 KRZANOWSKAI-I., Sperm quality and q u a n t i t y in inbred lines of mice and their crosses, Aeta Biol. Cracov (Ser. Zool.), 5 (1962) 279-290. io KRZANOWSKAH., Inheritance of reduced male fertihty connected with abnormal spermatozoa in mice, Acta biol. Craeov (Ser. Zool.), 9 (1966) 61-7o. i i KRZANOWSKAH., Factors responsible for spermatozoon a b n o r m a h t y located on the ¥ chromosome m mice, Genet. Res. Camb., 13 (1969) 17-24. 12 KRZANOWSKAH., Influence of ¥ chromosome on fertihty in mice, Proc. Int. Syrup. The genetics of Spermatozoon, (i972) 37o-386. 13 SLIZYNSKA H. AND B. 1V[. SLIZYNSKI, Cytologmal studies of sterile bulls with sperm head abnormality, J..dgr,e, Sci., 43 (1953) 253-255-

Absence of acrosome: an efficient tool in mammalian mutation research.

Spermatozoa without acrosomes ("acrosomeless") have been qualitatively and quantitatively analysed in several strains of mouse. The frequency of these...
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