Anat Embryol (1983) 168:445-453 Anatomy and Embryology �9 Springer-Verlag 1983

Glycogen Accumulations in Differentiating Mesonephric Ducts and Tubuli in Male Human Embryos

Lauri J. Pelliniemi 1, Pirkko Kellokumpu-Lehtinen 2, and Anita P. H o l l e r 3 Laboratory of Electron Microscopy 1, and Department of Anatomy 2, University of Turku, SF-20520 Turku 52, Finland, and Department of Anatomy 3 Harvard Medical School, Boston, MA. 02115, U.S.A.

Summary. Human mesonephric duct epithelial cells contained empty appearing regions in the infranuclear cytoplasm when prepared for trans- mission electron microscopy using glutaraldehyde and osmium fixation. The same regions stained positively with PAS in Epon sections for light microscopy suggesting that glycogen was present. Incubation with saliva abolished the reaction. For electron microscopy the glycogen stained very intensely if a mixture of osmium tetroxide and potassium ferro- cyanide was used instead of osmium alone. Glycogen accumulations were present between the ages of 5 to 10 weeks and absent at the age of 15 weeks. Reports by others indicate that glycogen may be present in different reactive forms in relation to its staining behaviour after various fixatives. The present results, and similar studies in other tissues, indicate that osmium tetroxide-potassium ferrocyanide fixative should be used routinely for preservation of embryos and fetuses and where indicated, for ultrastructural identification of glycogen and cytoplasmic filaments in clinical specimens.

Key words: Electron microscopy - Mesonephric duct - Fetus - Glycogen - Human development

Introduction

Differentiating cells in human blastocyst and in human embryonic and fetal genital systems contain accumulations of glycogen-like material (Hertig et al. 1958; Fukuda 1976; Niemi and Ikonen 1965; Pinkerton et al. 1961; Wartenberg et al. 1971). Similar accumulations have not been seen in animal gonads (Eddy 1974; Gondos and Conner 1973; Zamboni and Merchant 1973). On the other hand, large glycogen accumulations have been observed in the epithelial cells of mesonephric duct, which differentiates into epididy-

Offprint requests to: Lauri J. Pelliniemi, M.D., Laboratory of Electron Microscopy, University of Turku, Kiinamyllynkatu 10, SF-20520 Turku 52, Finland.

446 L.J. Pelliniemi et al.

mis in ram (Tiedemann 1971), but not in rat (Flickinger 1969) or man (De Martino and Zamboni 1966; Gould and Bernstein 1979; Holstein 1969).

We report here for the first time the presence of glycogen in human embryonic and fetal epithelial cells of the mesonephric duct. This material is not preserved by standard glutaraldehyde and osmium fixation, and there- fore may not have been visulaized in earlier studies.

Materials and Methods

The mesonephric ridges containing the mesonephric tubules, the mesonephric ducts, and the paramesonephric ducts were collected in the region adjacent to the gonad from 14 male human embryos and fetuses obtained from clinical interruptions of pregnancy. The ovulation ages were determined according to Streeter (1920) from tile measured foot lengths (FL) or crown- rump lengths (CR). The ages and corresponding lengths ranged from 5 weeks (CR 12 ram) to 15 weeks (FL 9.1 mm, CR 135 mm). The sex was identified by X chromatin staining and Y chromatin fluorescence before gonadal sex differentiation (Pelliniemi 1972) and by histologi- cal examination of gonads in older fetuses.

The ridges with the ducts were cut into 1 mm long pieces and immersed in a fixative containing 0.24 tool/1 glutardialdehyde (Glutaraldehyde EM, Leiras OY, Finland), 4.1 mmol/1 Ca C12, and 0.1 tool/1 sodium cacodylate-HC1 buffer (pH 7.4) for three hours at +4 ~ (Petlin- iemi 1975a). After washing overnight in 0.23 mol/1 sucrose, the tissue pieces were postfixed with 39 mmol/l osmium tetroxide in either 0.2 mol/l s-collidine-HC1 buffer (pH 7.4) or in 0A tool/1 s-collidine-HC1 buffer (pH 7.4) containing 36 mmol/1 potassium ferrocyanide (Kar- novsky 1971), both at 4 ~ for 2 h. The specimens were dehydrated in ethanol and embedded in Epon 812 (E. Merck, Darmstadt, FRG). Cross-sections of ducts were cut on a Sorvall MT 2 ultramicrotome with a diamond knife and stained with toluidine blue for light microscopy and with uranyl acetate (Watson 1958) and lead citrate (Venable and Coggeshall 1965) for electron microscopy. The specimens were examined with Philips 200 and JEOL JEM-100 C electron microscopes.

Glycogen was demonstrated by the periodic acid-Schiff (PAS) reaction after removal of the plastic from 1 gm sections (Lane and Europa 1965). Control sections were incubated with saliva prior to the PAS reaction for 30 min at room temperature.

Results

The epithelial cells of the mesonephric duct contained large clear areas in the infranuclear cytoplasm (Fig. 1) when fixed with conventional glutaral- dehyde and osmium tetroxide. These areas were not empty, however, be- cause they stained positively with the PAS reaction (Fig. 2). If the sections were preincubated with saliva, the PAS reaction was abolished. When a mixture of osmium tetroxide and potassium ferrocyanide was substituted for osmium tetroxide alone, the material in the previously clear areas was positively stained.

Electron microscopy showed that the basal areas of epithelial cells in osmium-fixed material were stained very weakly in comparison to the rest of the cytoplasm (Figs. 3 and 5). Osmium - potassium ferrocyanide fixation revealed accumulations of 30-50 nm glycogen particles with globular sub- units in the weakly staining areas (Figs. 4 and 6). These glycogen accumula- tions were most prominent between the ages of 5 to 10 weeks of prenatal development, and disappeared by the age of 15 weeks (Fig. 7).

Glycogen in Human Mesonephric Duct 447

Fig. 1. A survey light micrograph of mesonephric duct in cross section from an eight-week-old fetus. Glutaraldehyde and osmium fixation, toluidine blue staining, x 550

Fig. 2. Light micrograph of a mesonephric duct in cross-section stained with periodic acid - Schiff (PAS) reaction for the demonstration of glycogen. Large deposits of PAS-positive material (arrows) are present predominantly in the basal portions of the epithelial cells in the mesonephric duct. An eight-week-old fetus, glutaraldehyde and osmium fixation, x 550

Similar but smaller accumulations of glycogen particles were also ob- served, albeit less frequently, in the proximal mesonephric tubuli, the pri- mordia of the ductuli efferentes. The epithelial cells of the paramesonephric ducts, which do not degenerate until after the tenth week, contained relative- ly little glycogen.

Discussion

Accumulations of glycogen-like material are found in a great variety of epithelial tissues during prenatal development (cf. Table 1). After conven- tional aldehyde and osmium fixation, glycogen areas have an empty or a faint cotton-wool appearance. In these cases glycogen is not preserved with the fixative appropriately enough to bind subsequent heavy metal stains. A common feature in the differentiating cells is that glycogen gradual- ly disappears as the cells mature. However, in human epididymis, the basal cells contain glycogen in the adult period (Montagna and Hamilton 1952).

Ultrastructural and, in some cases, histochemical features are the criteria currently used to identify glycogen in tissue sections (Table 1). Biochemical studies and X-ray microanalysis indicate that osmium - ferrocyanide fixa- tives stain glycogen particles selectively (de Bruijn 1973), the binding takes place through the C2-C 3 dihydroxyl groups in glycogen (de Bruijn and den Breejen 1976), and Os-Fe complex is present in these particles (de Bruijn and van Buitenen 1980). The mechanism by which glycogen is stained in- volves chelation of the osmium - ferrocyanide complexes by donor atoms in the tissue macromolecules which, in turn, facilitates the reduction of osmium in sire to a more stable lower oxidation state and results in more osmium deposition than post-fixation with osmium tetroxide alone (White et al. 1979). A different view on the staining mechanism has been proposed

Fig. 3. Electron micrograph of mesonephric duct epithelium from a five-week-old embryo. Large non-staining areas (G) are evident in the basal portions of the cytoplasm. N nucleus. Glutaraldehyde and osmium fixation, x 3,300

Fig. 4. Port ion of mesonephric duct from an eight-week-old fetus. Glycogen accumulations (G) are seen in the basal cytoplasm. Glutaraldehyde and osmium - potassium ferrocyanide fixation, x 3,300

Fig. 5. Basal portions of mesonephric duct cells with large non-staining areas in the basal cytoplasm (G). N nucleus. An eight-week-old fetus, glutaraldehyde and osmium fixation. x 12,000

Fig. 6. Basal portions of mesonephric duct epithelial cells with large accumulations of glycogen (G). From a ten-week-old fetus. B basal lamina; N nucleus. Glutaraldehyde and osmium - potassium ferrocyanide fixation. • 12,000

450 L.J. Pelliniemi et al.

Fig. 7. Epithelial cells of the mesonephric duct from 15-week-old fetus. No accumulations of glycogen are present. B basal lamina; N nucleus. Glutaraldehyde and osmium potassium ferrocyanide fixation, x 7,100

by Rybicka (1979; 1981) who suggests that in canine cardiac muscle, the so-called glycogen granules contain protein rich in sulfhydryl groups rather than glycogen alone and that the granules visualized by staining with os- mium - ferrocyanide represent the proteinaceous component of glycosomes (protein-glycogen complex).

A review of papers related to the present work (Table 1) demosntrates the variety of glycogen staining in various cells. Two main types of reactions are seen. In most cases, glycogen is not stained after routine aldehyde and osmium tetroxide fixation but is stained with aldehyde and osmium - potas- sium ferrocyanide (Refs. 1, 2, 12, 13, 20 in Table 1). In other cases, glycogen is stained after routine aldehyde and osmium tetroxide fixation alone (Refs. 8, 11, 14, 15 in Table 1).

Variations in the staining of glycogen in the mesonephric duct have been observed in various species and at different stages of development. Using post-fixation with osmium - potassium ferrocyanide, we have demon- strated glycogen in the human fetal mesonephric duct and tubule for the

Tab

le 1

. A

ppea

ranc

e of

gly

coge

n-li

ke m

ater

ial

afte

r va

riou

s fi

xati

ons

Spe

cies

S

tage

T

issu

e or

org

an

GA

%

OsO

4 G

lyco

gen

OS

O4%

+

%

+ P

FC

(F

A %

) pr

esen

t st

aine

d

Gly

coge

n st

aine

d R

efer

ence

�9

%

hu

man

fe

tal

mes

onep

hric

duc

t 2.

4 1

+ -

1 +

1.5

hu

man

fe

tal

mes

onep

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tub

ule

2.4

1 +

- 1

+ 1.

5

hu

man

fe

tal

mes

onep

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duc

t 4

2 (-

) ( -

)

hu

man

fe

tal

mes

onep

hric

duc

t 1

(+)

( -

)

hu

man

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tal

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duc

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1 (-

) (-

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hu

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mes

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( -

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hu

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man

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tal

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cyte

s an

d 6

1 +

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erm

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onia

hu

man

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erm

cel

ls

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(5)

1 +

+

rat

embr

yoni

c ge

rm c

ells

2.

5 2

(-)

(-)

mou

se

embr

yoni

c pr

imor

dial

ger

m c

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2.

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- -

pig

embr

yoni

c pr

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dial

ger

m c

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2.

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pig

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l sp

erm

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onia

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1. P

rese

sent

stu

dy J

983

2. P

rese

nt s

tudy

198

3 =

3. G

ou

ld a

nd B

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tein

197

9 o

4. D

e M

arti

no

and

Zam

bo

ni

1966

5. H

olst

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1969

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1969

7. H

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1969

8. H

olst

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1969

9. H

offe

r 19

76

10.

Fli

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ger

1969

11.

Tie

dem

ann

197

1

12.

Faw

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and

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m 1

974

13.

Faw

cett

and

Dym

197

4

14.

War

tenb

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et a

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971

15.

Fu

ku

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1976

16.

Edd

y 19

74

17.

Zam

bo

ni

and

Mer

chan

t 19

73

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Pel

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a, 1

976

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b

(+),

(--

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t m

enti

oned

in

the

pape

r, b

ased

on

pres

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auth

ors'

eva

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452 L.J. Pelliniemi et al.

first time. The non-staining areas shown in the human mesonephric duct by de Mart]no and Zamboni (1966, Figs. I and 2) also suggest the presence of glycogen but the authors do not mention it in the text. Using only osmium tetroxide as a post-fixative, glycogen is not observed in the mesonephric duct of 1,2- and 16-week-old fetuses (Holstein 1969; Gould and Bernstein 1979) or in the fetal rat epididymis (Flickinger 1969). By contrast, a compre- hensive analysis of the mesonephric duct in the ram reveals glycogen accu- mulations similar to those in the present study (Tiedemann 1971). However, the glycogen deposits in the ram mesonephric duct differ from those in the human in that they require only aldehyde fixation and post-fixation with osmium rather than aldehyde and osmium - potassium ferrocyanide fixation as the glycogen in the human mesonephic duct.

The differences in the staining behaviour of glycogen in various cells suggest that glycogen is present as chemically different forms or complexes in various species and cells. On the other hand, the ambiguities in the inter- pretations of the reaction mechanism of the osmium potassium ferrocyanide fixative as described above hamper the selective application of this useful tool. Regardless of the exact mechanism of staining, it is clear that osmium - potassium ferrocyanide provides excellent preservation and contrast of glycogen complexes which in many cases otherwise remain unstained. Addi- tional contrast is also given to glycocalyx-related carbohydrate-containing structures, and cytoplasmic filaments (David-Ferreira and David-Ferreira 1980). Ferrocyanide-reduced osmium is therefore recommended as a routine fixative for the preservation of embryos and fetuses and where indicated, for ultrastrctural identification of glycogen and cytoplasmic filaments in clinical specimens.

Acknowledgements. The authors wish to express their appreciation to Dr. Don Fawcett for his constructive criticism of the manuscript and to Sirpa From, Georgia Hsiao, Mauno Lehti- m/iki, Marja Mattila and Ulla Mfintyl~i for excellent technical and secretarial assistance.

References

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Accepted November 7, 1983