BIOLOGY

OF

45, 493-497

REPRODUCTION

Seasonal

(1991)

Variations

in the Testis

(Lagostomus N. CARAVACA,3

L. B. FUENTES,3

Catedras

de Histologla

Instituto

maximus

y EmbriologIa,4

of Vizcacha

maximus)1

L. A. SCARDAPANE,3

L. E. PELZER,3

y Farmacolog(a,3

de Histologla

and Epididymis

R

S. PIEZZI,4

Universidad

Nacional

de San

Universidad

de Cuyo,

CONICET

Luis,

J. A. GUZMAN3

and

San

Luis,

Mendoza,

Argentina Argentina

ABSTRACT Seasonal

changes

rodent,

were

vicinity

of San

studies.

Significant

hibernal

period

in

Luis,

During

was

The

changes

complete.

These

activity

findings

relationship

activity

between

an

that

pineal

increased

in

increase

vizcacha

activity

and

highest

Many respond iological the

coming

season.

The

switching

on

and

cess

[1,21.

The

relation

between

male

is the induced

most pro-

changes

and

photoperiodic

gonadal

regression

to short days [5]. The present study was logical, tis and

morphometric, epididymis

maximus

maximus)

the

relationship The vizcacha

systems. The [61. The

May

September

‘This

research

was

de

San

2Correspondence: QuImica,

Bioquimica

Luis,

matings

occur

at other

in March

have a second in the male

or April, and that 36% pregnancy vizcacha

later is not

of fe-

in the year. well under-

of the

year, vizcachas

study

male

would

of environmental

AND

vizcachas

appear

to pro-

regulation

weighing

the histo-

latitude, 760 m altitude) 1986 to March 1988).

and

In San Luis, summer

and immediately days

have

14 h of light and an av-

at dusk

to feed

is 5.5 mo, and March-April

is the

h, and the average is 107 mm during

temperature is 10#{176}C. The average summer and 10 mm in winter.

Morphometric by and

the

Consejo

Secretaria

Argentina.

RS.P.

Dr.

Jorge

A. Guzmin,

and

y Farmacia,

Universidad

de

Investigaciones

de Ciencia JA.G.

are

Citedra Nacional

y Tecnologia,

members de

San

Luis,

Facultad 5700

San

is 10 rainfall

Studies

Body weight was measured to the nearest 0.1 g, and the testes and epididymides were weighed (sensitivity,0.01 mg). The right testis, right epididymis, and pineal gland were

y

Universidad

of CONICET.

Farmacologia, de

Cientificas

4-7

decapitated (March

of 25#{176}C. In winter, the light phase

and

of re-

METHODS

(4 animals/mo

erage temperature

at dawn

that per

kg) were used in the study. The animals were killed by shooting, at night, in their natural habitat near their burrows in the vicinity of San Luis, Argentina (330 20’ south

1989.

supported

A possible

cycle.

burrow

emerge

Argentina,

A gradual

hydroxyindole.O.

reproductive

MATERIALS

1991. 22,

an annual

begins

periods

Ninety-six

changes in the tesvizcacha (Lagostomus photoperiods

significant

spermatogenesis

testosterone. pineal

season-although

vizcachas Reproduction

some

this response

to examine

seasonal

breeding

to short pho-

of photoperiod to pineal activity. lives in large colonies in extensive

17,

T#{233}cnicas (CONICET), Nacional

in different

gestation period

Received

designed

and biochemical of the adult male

animals

Accepted

by exposure

of the pineal prevents

2)

suggested.

vide an interesting production.

studied of these speauratus), in which

(Mesocricetus

is induced

[41. Removal

toperiod

of the most

the

of

in August. with

and

to secrete period,

a short decrease

stood. According to Llanos and Crespo [6], vizcachas are fertile throughout the year in their natural habitat. Because of their particular habitat and short exposure to light in

the sexual cycle of mammals has long been recognized [31. The pineal gland has also been implicated in the regulation of seasonal reproductive cycles in several photoperiodic mammalian species. One cies is the Syrian hamster

pronounced

the

in winter.

year, which

off of reproduccycle

America),

and

increased

in

structural

times of the year [7]. Llanos and Crespo [61 reported vizcachas in the wild have only one breeding season

mammals that are seasonal breeders are able to to annual changes by adaptive alterations in physas well as in behavioral status in anticipation of

tive function during the annual breeding striking example of such a photoperiodically

ability

exhibits

is also

and

observed, was

in this

observed

main

INTRODUCTION

most

weight

as in the

conditions

regression

in South

American

burrows

of spermatogenesis,

regression

gonadal

their

for biochemical

(winter

arrest

with

near

night

used

In July-August

Conversely,

values natural

and

a South

maximus),

the

of spermatogenesis-was

well

as

winter).

under

removed

period,

season,

activity

during

weights,

arrest

(early

gonadal

1)

this

In this

sexual

to the

adult

were

found.

period-without

(autumn).

maximus

decapitated

epididymal

during

in June-July

male

and glands

were and

regressed

in contrast

the

pineal

testicular

(Lagostomus

vizcacha

killed

cycles

in

a recovery

decreased

indicate

seasonal

gonads

appeared

male

were and

April-May

indicate

results

transferase

Our

The

during

activity

of testicular

methyl

with decline

observed.

adult

animals

epididymides,

(spring),

of gonadal

reduction

testes,

quiescence,

were

in the

2 yr, the

associated

of sexual

September-November

expression

activity for

Monthly,

Argentina.

testosterone

serum

reproductive

investigated.

de

quickly removed and placed in vials on dry ice. The vials were then sealed, kept frozen, and shipped to the labora-

Luis,

Argentina.

493

494

FUENTES

tory

for

biochemical

assays.

of blood from the terone determinations. were

fixed

trunk

in Bouin’s

fluid

tology. Structural stereological testes and epididymides August, and October selected

according

weights

and

minimal,

and

amined

by

scope.

(4

up

samples

serum testosleft epididymis for

routine

his-

Cross

were

during

sections

Orthoplan

taken

Leitz

at random

printing.

ex-

at 25 X and

A total

of five

pho-

amined in a Bitpad 1 (Summagraphics, Co., Hartford, CT) digitizing tablet and processed according to Peachey [8], with a morphography program in a Hewlett-Packard (Palo Alto, 150 computer. testes were

Data were

assessed

subjected

established

by Johnston

Biochemical Serum

and

Zucker

concentrations

determined

by

use of specific antibody against testosterone-3-O-carboxymethyloxime-BSA with the following cross-reactivities: 0.2% with cholesterol, cortisol, corticosterone,

progesterone,

trone, pregnenolone, dehydroepiandrosterone, one, and estrone; 71% with dihydrotestosterone; androstenedione; 0.6% with with 3I3,173-diol-5a-androstane; androstane;

and

0.4%

previously described The pineal glands sayed

individually transferase

[‘4C]Methyl-S-adenosylmethionine, chased from New England Nuclear scintillation spectrometer (Beckman Palo Alto, CA) was

used

tosterone and HIOMT and August 1987.

androster0.7% with

313,1713-diol-5a-androstane,

[11]. were

for hydroxy-O-methyl

es-

313,1713-dio-5a-androstane; 2.4% 4.8% with 313,17P-diol-5a-

with

to measure activity

were

weighed (HIOMT)

and

Epididymal

and

I-.

Ui

> -

Ui

Ui

a. JFMAMJ

JAS0NDJ

FMAMJ

.1 ASON

FIG. 1. Annual variations in the weights of the right testis and epididymis for the adult male vizcacha. The breeding period is indicated by the cross-hatched bar. Values are means ± SEM from 4 animals in each month. Statistical analysis was performed by ANOVA: A,’ B (p < 0.05), B C (p

as

54 mCi/mmol, was pur(Boston, MA). A liquid LS-233, Beckman lnstr., determined

ILit

(autumn). These maximal values were followed by a rapid and significant (ANOVA; p < 0.05) decrease in July-August. Redevelopment began in September (late winter and early

[10].

were

-J

by rating

Studies testosterone

-

to ANOVA.

histophysiologically

slides of 4 animals by month according to the method described by Grocock and Clarke [9]. A spermatogenic index was calculated on the basis of inspection of ten slides per testis. Numerical values were assigned according to the criteria

0

15

a..

cross of ep-

ididymal duct were examined per photograph. To estimate the tubular surface of testis epididymis, the prints were ex-

The

-

#{149} -

I-

micro-

tographs were used for each animal. About 10-15 sections of seminiferous tubules and 6 cross sections

CA) HP

2.0

C), -‘C

gonadal

(maximal,

in an were

of the

characteristics

activity).

100 x

performed using the killed during April, The samples were

variations

histological

microscopy

to

and

processed

seasonal

intermediate light

and

for

AL.

0

to the

their

arterial-venous

studies were of vizcachas animals/mo).

Photomicrographs

enlarged

Mixed

were collected The left testis

Er

relwere

gust; in April and October, height were greater than tertubular connective

tubule in August,

tissue.

diameter and epithelial and there was less in-

SEASONAL

FIGS. 2 to 9. Paraffin FIGS. 2 and 3. Testis Spermatozoa are present

sections (6 m) of seminiferous and epididymis from a vizcacha in the lumen. x250.

GONADAL

tubules and killed in April

CHANGES

epididymal (autumn).

IN MALE

ducts Arrow:

VIZCACHA

of vizcachas. Stained with hematoxylin and eosin. cluster of Leydig cells. Note height of the epididymal

495

epithelium.

FIGS. 4 and 5. Testis and epididymidis of a vizcacha killed in October (spring) showing gradual recovery of gonadal histology. x250. FIGS. 6 and 7. Testis and epididymidis of a vizcacha killed in August (winter). Seminiferous cords are composd predominantly of Sertoli cells (Fig. 6), and reduced epididymal ducts together with increased insterstitial tissue (Fig. 7) are the main histological characteristics. x250. FIG. 8. Higher magnification of the testis of a vizcacha killed in December (summer). Note complete spermatogenesis in the seminiferous tubules and a cluster of well-maintained Leydig cells (arrow). x600. FIG. 9. Testis of vizcacha killed in August (winter). Cluster of reduced Leydig cells showing small, dense nuclei (arrow) and seminiferous cords. x600.

FUENTES

496

>( LU

a z

Er

AL.

25% for seminiferous Middle values were

tubules obtained

Serum

and

Testosterone

The

4

general

parameters (-)

z

3

0

2

0

than

4 Ui

a:

of

was

function.

in Figure

ducts.

HIOMT

results

repeated

for

In effect,

1 and show

in August. The values for pineal HIOMT

other

a correlation

gonadal weights and serum testosterone was observed (Fig. 11). The results were

to those shown

I-

Pineal

pattern

of reproductive

between trations

w

and 83% for epididymal in October.

concencoincident

higher values in May

of the vizcachas

are

also

shown in Figure 11. In contrast to the results for testosterone levels, the HIOMT activity was lower in May than in August.

0

V)

I0IJIFIMIMIMIJIJIAISI0INI0I FIG. 10. Spermatogenic index showing seasonal variations. The index was calculated from the inspection of ten slides per vizcacha. Each column represents the mean of 4 animals. Numerical values were assigned according to the following criteria for stages: 5, the seminiferous tubules were large and spermatogenesis was complete; 4, spermatogenesis was complete but spermatozoa and elongated spermatids were decreased in number; 3, the number of spermatozoa was further reduced, and elongated spermatids were present; 2, elongate spermatids were absent; 1, only Sertoli cells, spermatogonia, and primary spermatocytes were present; 0, only Sertoli cells and spermatogonia were detectable (according to Grocock and Clarke 191).

DISCUSSION The tive

results

activity

the annual vironmental

Figure

10 shows

the spermatogenic of the

index

the four seasons

genesis autumn

(stages 4 and 5) was detected during months. In contrast, spermatogenesis

plete during A dramatic August.

and beginning activity was

seasonal periods conditions. This period

nesis

followed

during

Analysis

(Table

during

using

vizcachas

LU > LU

of seminiferous were performed and

of surface in August, compared

1. Seasonal and surface

variations areas.

in testis

Uo

2 a

li-i

October.

A

LU I-

0 I-. >-

Months April August October *Values

± ± ±

0.30 0.07 0.24

are expressed

8.800

±

2.188

±

4.635

±

as means

In

300 200

of

Epididymis

Tubular surface (1J.m2)

Tw/Bwt 1.47 0.25 0.66

comparison

400

-

6

100

Testis

0.8(a) 0.3(b) 0.9(c) ±

±

0.04

±

0.66

±

SE. Four animals

Statistical analysis was performed by ANOVA: (a) (b) (C) (p < 0.05) (d) (e) # (f) (p < 0.05). tTw/Bw: testicular weight/body weight. Ew/Bw: epididymal weight/body weight.

0#{149}

Ductal surface (m2)

Ew/Bw* 0.18

0.07 0.01 0.24 were

of

3

C

0

to April values, was

and epididymis:

expression

autumn.

5

-

> TABLE weights

and

4

correlation between both weights and stereological quantification was observed. On the basis of these data, the reduction

by a significant

summer

6

-I

LU

q,

killed in April, August,

of

and may be regulated by enannual cycle is characterized

-J

U)

analyses surfaces

reproduc-

(I.)

of spring). observed in

1)

structural stereological and epididymal ductal

that

as a function

spermatosummer and was incom-

u’)-

The tubules

indicate is cyclic

of gonadal quiescence and arwhich is most pronounced in Auperiod without arrest of spermatoge-

spring,

activity

L-

Moi’phometric

study

vizcacha

registered

year. Complete

July-October (winter arrest of spermatogenic

present

male

by a short hibernal rested spermatogenesis, gust, and a recovery gonadal

during

of the in the

16.588 2.770 18.051 used

± ± ±

1.3(d) 0.80(e) 0.76(f)

by month.

0

I Q.

I JIF

IMIA

AUTUMN

IMIJ

I

WINTER

lilA

I 1510

INIDI

FIG. 11. Changes in the serum testosterone levels and pineal HIOMT activity of adult male vizcacha. Values are means ± SEM and the number of experiments are indicated within the bars. The asterisks indicate a statistically significant difference (p < 0.001, “p < 0.01) between the means of May and August (Student’s t-test).

SEASONAL

The occurrence of spermatogenesis, cells, and the size/weight/surface organ

for

male

hormone

sex

hormone)

secretion

borne From

when

of the

suggest

day

length

GONADAL

the enlarged epididymis

CHANGES

Leydig (target

increased

VIZCACHA

sex

this

was

out by the results of the assay of plasma testosterone. these results, we conclude that spermatogenesis in

in winter

than

testis,as well as gametogenic tuations according in spring and the endocrine

activity,exhibits rhythm

to season. The observed

testosterone and in Leydig

changes

The

authors

in summer.

for their

technical

manuscript.

a seasonal

to be

participation of

These

parameters

duration

may

suitable

1. BakerJR, agrests).

sexual

is rodent

riods of light [6].These

may be winter.

vizcacha

condition

normally animals

because

an important During spring

is gradually

6. Llanos

make

to twilight (crepuscular is more

the

light)

prolonged.

in different

mammals

may

induce

j Reprod

8. Peachey

W.

13.

[5, 18, 19]. testicular

The

regression

gonadal pineal during

funcgland

to pineal

influence.

Consequently,

testicular

of

winter

and

Acred

for typing

the

appreciated.

of the field

after

mouse

(Micrortat

BJW, Jorgenson Press;

CB (eds.),

1968.

sexual

cycles.

Relations

November

and

December.

control

of

15.

of ferrets Proc

of

R Soc

hamster

testis.

Science

1967;

epi-

de

Ia vizcacha

Ia provincia

Fertil

de

A simple

control

of reproduction

in

(Lagosfomua

Entre

Rios.

maximus

maximua

Invest

Revista

Agric

Bs As

digital

1974;

Lofts

B. Animal

de

control

Lagactomus

for electron

L, Foglia

diabetic

max-

microscopy.

in the vole,

Control

UI.

Microtus

development

Reprod EH.

Physiol

Ut

1979;

J Biol

function

1976;

26:387-394.

transferase

Chem

of Biology’s

and

21:1-8.

Endocrine

Am

of hydroxy-O.methyl

lighting.

In: Institute

1975:

gonadal

Biol

V. Charreau

rats. Acts

SH.

photoperiodism.

P. 5-Methoxyindoles

activity on

bispidus.

by environmental

1965;

Studies

ac-

240:949-955. in Biology,

vol.

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In: Mess

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Reiter RJ. The pineal assessment. In: Gupta

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Valle

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JR Photoperiodic

M, Biella

Axelrod

physiology

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seasonal

L, Pevet

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P (eds.),

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The

1985:

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Current

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gland pubertal development in mammals: a state of the ID, Reiter RJ (eds.), The Pineal Gland during Development.

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20.

de

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JA. Ecologia

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reproductive

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10. Johnston

In

(short day lengths). As spring and summer approach (long day lengths), the gonads become active and are unresponsive

Diaz

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17.

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pineal

Crespo

imus.

diating

information

added

M. Photoperiodic

en el nordeste

9. Grocok

for gonadal involution summer (14L: 1OD), the

daylight period

AC,

agrestfc.

where

York:

of mammalian light

Endocrine

tramicrotomy

to long pe-

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RJ. The

7. Weir

16.

the vizcacha

Mu#{241}ezde is much

In: Barrington

New

Modification

to electric

S, Menaker

1952;

again activated in the presence of favorable conditions. It is known that the pineal gland has an essential role in metion

Lilia Ricur

Juan

110:332-348.

Blainv.)

at a given time, but and

TH.

mammals.

these seasons, vizcachas become refractory to the inhibitory effects of photoperiod, and the reproductive system is

photoperiod

Mrs.

activity.

in Endocrinology.

sexes

1932;

reproduction

season [17].

of testicular

B. Pattem

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proxi-

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158:925-928.

temperature is about 18#{176}C. The animals emerge from their burrows during the period of darkness. This natural condition during

We

assistance

his valuable Sarmiento

Ransom RM. Factors affecting the breeding Part 1: Light. Proc R Soc 1932; 110:312-322.

Perspective

5. Reiter

of seasonal

also to actually anticipate the upcoming the necessary physiological adjustment The vizcacha

for Fogal

REFERENCES

the reproductive as occurs in other

remains

regulation

be consid-

living in their natural habitat [15, 16]. Depenphotoperiodic fluctuations allows animals not only

in the

Charreau

to Teresa

fluc-

2. Lofts

ered as important variables that control processes of mammals [13, 14]. However, the

linguistic

Eli.

and

in serum

4. Gaston

natural photoperiod.

for

to Dr.

facilities

collaboration.

The

and

photoperiodic

grateful

laboratory

cells (smaller in winter and larger

summer) suggest activity.

stimulus

are

for providing

3. Bissonette

of animals dence on

during winter have cytological secretory activity than in summer the pineal HIOMT level is higher

ACKNOWLEDGMENTS

This discontinuous gonadal activity can be attributed to seasonal changes of different environmental factors, such as ambient temperature, food availability, rainfall periods,

rodents,

by dem-

period. A similar biological pattern has been

observed in the majority of seasonally breeding mammals [13]. It is not surprising that the endocrine component of the

mate

This hypothesis is supported study made by us [20], which

onstrated that pinealocytes characteristics of a higher and autumn. Coincidentally,

vizcachas is not continuous. It has an important climax during summer and the beginning of autumn, coincident with the breeding

497

didymal activity increases. an electron microscopic

male

is decreasing;

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nervous

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4:211-219.

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