Acta hlstochem. 70, 22-30 (1982)

Laboratory of Alllmal Endocrlllology and Tissue Culture,

Department of Alllmal PhysiOlogy, Institute of Zoology, Jagiellolllan Ulllver~Ity, Krak;ow, Poland

Histochemical evaluation of L1fi,3/J-OHSD activity in two types of

porcine corpora lutea and granulosa cells in tissue culture

By EWA GREGORASZCZUK and ANNA WOJTUSIAK

WIth 7 figures

(Received AprIl 9, 1981)

Summary

ActIVity of Ll5,3,B-hydroxysterOld dehydrogenase (Ll5,3·0HSD) III cultured porcllle corpus luteum cells of early and mIddle luteal phase and granulosa cells was histochemlCallllvestigated.

Control cultures as well as cultured with additiOn of luteIlllzmg hormon (LH), prolactm, human choriOlllc gonadotrophm (HOG), and estradiOl 17,B were carried on.

The stImulatory mfluence of LH and HCG on 3 cell types has been stated. Prolactm and estra· diOl stimulated only the cells of corpus luteum from early luteal phase.

Introduction

An important role m the biosynthesIs of steroid hormones is played by L15,3/J­hydroxysteroid dehydrogenase (L15,3{J.OHSD). Its actiVIty was studied m the steroid hormones synthetizing cells by many authors. BJERSING (1967) studied the activity of steroid dehydrogenase in the porcme ovarIan folhcles and corpus luteum.

He found that the actIvity of this enzyme ohanges together WIth the secretory function and morphology of the oells of these organs during the cycle. Similar studies were done on the folhcles and corpus luteulll of the cow. High aotivity of L15,3/J-OHSD was found in the cells of the porcine ovarian follicle and corpus luteum in the different stages of the development of these organs.

KENNY (1964), LOBEL and LEVY (1967), ZAMORSKA (1972), STOKLOSOWA et al. (1978) found that there were clear differences between granulosa cells and theca interna cells in the level of L15,3/J-OHSD activity both III the suspensIOn of isolated cells and in culture. The aim of present work was:

1. To study the actIvity of L15,3/J-OHSD in the granulosa and theca mterna cells isolated from the corpora lutea which were in the early and middle luteal phase.

2. IdentifICatIOn of these 2 types of cells in cultures estabilished from the isolated cells of corpus luteulll.

3. To determine the relative importance of granulosa and theca interna in the formation of corpus luteUlll.

HIstochemIcal evaluatiOn 23

4. To study the influence of gonadotropic hormones and estradiol on the activity of Ll5,3p.OHSD in cultured cells.

5. To compare the results of the present research with those conducted with granu­losa cells.

Materials and methods

PIg ovarIes were collected from slaughterhouse ammals. For further studIes ovarIes whlCh

were m 3 followmg phases of the cycle were chosen: m the follIcular phase, postovulatory phase,

and mIddle luteal phase. The phase was determmed accordmg to AKINS and MORRISSETTE (1954).

Granulosa cells were Isolated usmg the method of CHANNING (1966, 1969) and the cells 01 corpus luteum were Isolated accordmg to the method of GRYGON and STOKLOSOWA (unpublished).

The suspensiOn of the Isolated cells were submItted to the hlstochemlCal test for the actIVIty

of Ll5,3P-OHSD FISCHER and KAHN (1972) using dehydroepiandrosterone (DHA) as substrate,

(NAD) as cofactor, and mtrotetrazohum blue (NBT) as hydrogen acceptor. Cells whlCh showed the

presence of forma zan granules after the reactiOn were ,dent,hed as sterOldogemc ones. The reactlOn

was c1asslhed as strollg ( + + + ), intermedIate (+ +), or weak (+) dependmg on the mtenslty of

colour. Tho percentages of cell classes WIth dIfferent enzyme activIty present m the cell suspensiOn was depIcted on the hIstogram (fig. 1). The cells were also classifIed accordmg to then SIze. Cells

d,ameter was measured and the frequency of dIfferent SIze classes m the cell suspenslOn was com­

puted.

Cells were cultured m MedIum 199 supplemented WIth 10% of calf serum (Laborytory of Sera and Vaccmes, Lublin, Poland). After IsolatlOn granulosa cells were dIluted to the concentratlOn of

106 cells/ml and luteal cells to the concentratiOn of 3 to 5 X 105 cell/mI. The culture were set up either In medmm WIthout hormones control cultures or one of the followmg hormones was added:

10 or 100 ng/ml LH (NIH-LH-S 17 OVINE), 600 ng HCG BlOgonadyl (Laboratory of Sera and Vaccmes, Lublm, Poland), 100 ng/ml prolactm (OVINE BwloglCal Standards NatlOnale lnst.) or oestradlOl-17 p (Sigma) 150 ng/m!. The medmm was changed each second day and every tIme

the hormones were added. In the same mtervals 1 culture was termInated and approXImate

a half of the cells was studIed h,stochemlCally for the Ll5,3P-OHSD actIVIty. The other half was stamed WIth MAy-GRUNWALD-GIEMsA staIn to exalmne the morphology of the cells MERCHANT et a!. (1969). The cultures wore contmued for 10 days.

Results

Postovulatory corpus luteum (figs. 1, 2): The frequency distributIon of different cell types present m the suspension classified accordmg to their enzymatIC actiVIty or size IS depicted in (fig. 5). A small percentage of cells (12 %) showed no enzymatic activity i.e. the results of the reactIOn for the Ll5,3P-OHSD activity were negative. These cells were small with the dIameter of 10 /lm. Cells which showed a positive enzymatic reactIOn were varIable in SIze and their dIameter ranges from 10 to 30/lm. One could dIstingUIsh 4 dIfferent classes of these cells (fIg. 5a).

Only 2 cell types were found in cultures established from the cells Isolated from the postovulatory corpus luteum. One of them showed a hIgh activity of Ll5,3p-OHSD. The cells of another type showed a weaker actiVIty of thIS enzyme and grew concen­trically around the cells of the 1st type (fig. 5 b). The activity of Ll5,3P-OHSD was much higher in the cells cultured in the presence of 10 ng LH, HCG, or PRL (figs. 5 c, 5d) than in control cultures. The stimulatmg effect was the most clear m the case of

24

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·X

o UJ

f­Z a u

j --' UJ

U

,,0 o

EWA G;REGORASZCZUK and ANNA WOJTUSIAK

COR PUS LUTEUM CELLS

EARLY LUTEAL PHASE

Q ~ 0 '" 0

'" '" M

8 8 8 8 :3 CD ~ ~ I'l '" '"

COR PUS LUT E UM CE LLS

MIDDLE LUTEAL PHASE

~ '" '" :3 :3 CD 0

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0 ;- JJM :3

0 M

5 A .3~ OH SD

_ NEGATIVE REACTION

DpOSITIVE REACTION

FIg. 1. HIstogram of corpus luteum cells of early and middle luteal phase III suspension prIOr to

culture. The strIped columns show percentage of non-steroidogemc cells (-) ,15-a,B-OHSD. The whIte columns represent the percentage of sterOIdogemc cells which revealed actIvIty of the enzyme (+) ,15,a,B-OHSD. -

10NG 100NG

2

CORPUS LUTEUM CELLS OF EARLY LUTEAL

4 6 8 10 DAYS OF CULTURE

C~ WHOLE RECTANGULAR REPRESENTS 100% OF CELLS EXHIBITING VARIOUS ACTIVITY OF t,.5,3r-OHSD

-+++ 1ZZ2J++ t:J+

FIg. 2. DynamICs of the activIty of ,15,a,B-OHSD III corpus luteum cells of early luteal phase in

lO-day culture.

a lower dose of LH (10 ng LHjml). The stimulation was weaker in cultures with HCG or prolactin.

Oorpus luteum from the middle of the luteal phase (figs. 1, 3): Approximately 80 % of the cells isolated from the corpus luteum which was in the middle of the luteal phase showed a positive reaction for the L15,3/J-OHSD. 2 types of cells could be also

C I ]

Histochemical evaluatiOn

CORPUS LUTEUM CELLS OF MIDDLE LUTEAL PHASE

DAYS OF CULTURE

I WHOLE RECTANGULAR REPRESENTS ~------' 100% OF CELLS EXHIBITING VARIOUS

ACTIVITY OF f:::..5. 3r-OHSD

25

Fig. 3. DynamICS of the activity of Ll5,3p·OHSD III cultured corpus luteum cells of middle luteal

phase durmg lO·day culture.

GRANULOSA CELLS

C~

IWHOLE RECTANGULAR REPRESENTS ~------' 100 % OF CELLS EXHIBITING VARIOUS

ACTIVITY OF .c,5. 3 r- OHSD

Fig. 4. DynamICS of the activity at Ll5,3p·OHSD III cultured granulosa cells durlllg lO·day cultures.

distinguished among these cells: the 1st type constituted the cells which were 40 pm in diameter (50 % of all cells in the suspension), the 2nd type 25 pm m diameter was distinguishable by the dIffuse histochemical reaction for the Ll5,3,B-OHSD activity [30% of all cells III the suspension (fig. 6a)]. The 1st type of sterOldogenic cells was not found in the suspension of cells isolated from the postovulatory corpus luteum. The remaining 20 % of cells showed negative reaction for the Ll5,3,B-OHSD activIty. It is believed that they are non-steroidogenic cells (fig. 1).

The cells isolated from the corpus luteum whICh was in the middle of the luteal phase formed a homogenous monolayer in culture and looked hke typical luteal cells.

26 EWA GREGORASZCZUK and. ANNA WOJTUSIAK

Sa ..

.. Sb

Fig. 5a. SuspenslOn of corpus luteum of early luteal phase submitted to histochemical test for

Ll5,3p-OHSD. 4 cells types vIsible. Cells were classIfied accordmg to their size and mtenslty of histochemICal reactlOn. X 400.

b. ActiVity of Ll5,3p-OHSD m corpus luteum cells of early luteal phase, 4-day control culture. X 200.

c. Activity of Ll5,3p-OHSD m corpus luteum cells of early luteal phase 4-day of culture grown III

presence of prolactm. Very strong actrnty of dehydrogenase m majority of cells. X 200. d. ActIVity of Ll5,3P-OHSD III culture grown m presence of HCG. X 200.

HIstochemICal evaluatIOn 27

FIg. Oct. The suspenSIOn of corpus luteum cells of mIddle luteal phase submItted to hIstochemICal

test for ,15,3,B-OHSD. 2 cell types vIsIble: 1. very larger, showmg strong hIstochemICal reactIOn.

2. small ones revealmg much weaker actIvIty of the enzyme. X 400.

b. A monolayer of corpuH luteum cells showmg acttvity of the ,1',3/'1-0HSD after 4-day m control

culture. X 200. c. 14-day culture grown m presence of IOO ng LHjml. Atrong histochemICul reactIOn vISIble.

X200.

d. ActIVIty of ,15,3/'1-0HSD In cultured corpus luteum cells of mIddle luteal phase. 4·day culture in medIUm supplemented WIth estradIOl. X 200.

28 EWA GREGORASZCZUK and ANNA WOJTrSIAK

7a .....

~.

Fig. 7. a. 2·day monolayer of control granulosa cell culture showing actIvIty of Ll5,3,B·OHSD. X 200. b. Granulosa cells cultured for 2 days In medmm contammg 600 ng of HCG/ml. Cells assayed for activity of Ll5,3,B·OHSD. Very strong actIvIty of the enzyme IS VISIble. x200. c. ActIVIty of Ll5,3,B-OHSD m granulosa cells In 2-day culture In presence of prolactIn. X 200.

Hlstochenncal evaluatlOn 29

In the control cultures the activity of the LJ5,3,B-OHSD was weaker than control oulture from corpus luteum of early luteal phase (fig. 6 b). More pronounced aotivity of this enzyme was found III the cultures with gonadotropic hormones LH or HCG, but the stimulatmg effect was much weaker than in corresponding cultures of the early corpus luteum cells (Figs. 6c, 3). It was found that estradiol stimulates the activity of LJ5,3,B-OHSD III the cultured cells. This stimulating effeot of estradIOl was much weaker in oultures of the cells isolated from the postovulatory corpus luteum. LuteotroplC effect of estradiol was much stronger than the effect of prolactm, the reverse was true for the cells isolated from the postovulatory corpus luteum (fig. 6d).

Granulosa cells (Fig. 4); Only 1 type of cells was found m the suspension. The oells were small (15 ",m in diameter). The reaction for the LJ5,3,B-OHSD showed a weak activity of this enzyme. Granulosa cells attached to the glass surface m the first days of oulture, but the monolayers were formed not earlier than after 3 to 4 days of culture. The activity of LJ5,3,B-OHSD was relatIvely hIgh in control cultures (fig. 7 a). In the oultures with LH or HCG the activity of this enzyme was much stronger and did not decline until the end of culture (fig. 7 b). Prolactin stimulated the activity of LJ5,3,B­OHSD only weakly (fig. 7 c). No stnIlulatmg effect of estradiol was found (fig. 4).

Discussion

STOKLOSOWA et a1. (1978) found that Isolated folhcular granulosa cells and theca Interna cells could be dlstmgmshed m the suspenslOn by the actIVIty of ,15,3P-OHSD. The actIVIty of thIS en­zyme was weak In granulosa cells and much stronger m the theca mterna cells. ThIS fmdlng was

accepted as a baSIS for our study and we hoped to follow the fate of granulosa and theca Interna cells m the corpus luteum. It have been found that approxImately 40 % of cells Isolated from post­

ovulatory corpus luteum had a SImIlar dIameter and ,15,3P-OHSD actIVIty as granulosa cells. 18 % of cells could be dlstmgmshed by theIr SIze and enzymatIC actIVIty as theca cells. The remammg class of cells m the suspenslOn comprIsed larger cells whrch most probably were luteIlllsed granulosa and theca cells (fig. 5 a). Granuloea lutem and theca lutem cells were also observed by ZAMORSKA (1972).

:\IorphologICal and functlOnal differentiatlOn of cells wa,; also preserved m culture of cells Isolated from postovulatory corpus luteum. 1 type of cells showed a stronger reactlOn of ,15,3P­

OHSD, another one a weaker actIVIty ot thIS enzyme. Later In culture the dIfference between these 2 types ot cells dimmished a~ a result of luterlllzatlOn.

PorCine granulosa cells and theca Interna cells were studIed In VIVO hlstochemwally by ~IIRECKA

(1972). She found a conSIderable actIVIty of "J5,3P-OHSD m theca mterna cells and notICed a declm­

mg actIVity of thIS enzyme In the agmg corpus luteum. The decrease of ,15,3P-OHSD actIVIty In

theca cells of corpus luteum durmg ItS mvolutlOn connected wIth the degeneratlOn of luteal cells

was found also by ARATEI and BARBAROSA (1968). Our results lead to SImIlar concluslOn. The cells

Isolated from the corpus luteum whICh wa,; m the nuddle of the luteal phase showed weaker and

more homogenous actIVIty of ,15,3P-OHSD than the cells from younger corpus luteum. It IS found

also thdt prolactm does not stImulate the productlOn ot progesterone In granulosa cells cultured

in vitro (GREGORASZCZUK, unpublIshed). On the contrary a conSIderable stllnulatmg eife('t of pro­

lactm as measured by the actIVIty of ,15,3P-OHSD on cultured luteal cells Isolated from postovula­tory corpus luteum was found. Most probably thIS IS due to the presence ot thecal cells m post­ovulatory corpus luteum. STOKLOSOWA et a1. (unpublIshed) found that prolactm ilnd estradlOl stlllluldted the secretIon of progesterone m theca cells.

30 EWA GREGORASZCZUK and ANNA WOJTUSIAK, HIstochemIcal evaluatIOn

Acknowledgements

The authors wish to thank to Prof. Dr. S. STOKLOSOWA for her mterest and stImulatmg sugges­

tions and to the WHO for the SSP support. Th,s mvestigatIOn was supported by PolIsh Academy

of Sciences, Dept. of Medical SCIences, grant A-43, by MR II 10 from the AgrICulture Academy m

Wroclaw, and by fund. No. MR I 12.2.2.4.

Literature

AKINS, E. L., and MORRISSETTE, M. C., Gross ovarian changes durmg eRtrous cycle of swme. Amer.

J. Vet. Res. 29, 1953-1955 (1968). ARATEI, H., and BARBAROSA, C., Geneza hormonilar ovarIem. H,stoch,mla SI bIOch,mla steroid-

3fl-ol dehidrogenazcI III ovarul de mamnllfera Sus scro/a. Stud. Cere. EmbrIOI. C,tOI. 5, 53 - 64

(1968). BJERSING, L., On the morphology and endocrllle function of granulosa cells III ovarIan follicles

and corpora lutea: BIOchemIcal, hIstochemical and ultrastructural studied on the porcme

ovary with ApeClal reference to steroId hormone synthesIs. Acta Endocrlllologica, Copenhagen,

SuppI. 125, 1-23 (1967). CHANNING, C. P., Progesterone bIOsynthesIS by equllle granulosa cells growmg In t,ssue culture.

Nature, London 210, 1266 (1966).

- Tissue culture of eqmne ovarian cell types. Cultures methods and morphology. J. EndocrmoI.

43,403-414 (1969a). FISCHER, T. V., and KAHN, R. H., HistochemIcal studleR of rat ovarian follIcular cells in vttro.

In VItro 7, 201-205 (1972).

KENNEY, R. M., HistochemICal and bIOchemICal correlates of the estrous cycle In the normal cyclIc uterus and ovary of the cow. Ph. D. TheSIS. Cornell Umv. New York 1964.

LOBEL, B. L., and LEVY, E., EnzymatIC correlates of development, secretory functIOn and regres­SIOn of follIcles and corpora lutea In the bovllle ovary. Acta endocrmoI., Copenhagen, Suppl. 132,1-63 (1968).

MERCHANT, D. J., and KAHN, R. H., Techmques and proeedures. In' Handbook of cell and organ

culture, chapt. 5. Burgess, Mmneapolls 1968. MIRECKA, J., Histochemical differentIatIOn between the cells of theca and granulosa orIgIn In the

porcme Graafian follICles and corpora lutea. FolIa Hlstochem. 11, 221- 234 (1974). STOKLOSOWA, S., BAHR, J., and GREGORASZCZUK, E., Some morphological and functIOnal charac­

terIstIcs of cells of the porcme theca mterna m hssue culture. BIOI. Reproduct. 19, 712 -719

(1978). ZAMORSKA, L., The actiVity of some mtracellular enzymes In the corpus luteum spurmm of the

domestIc pIg Sus scro/a domestica L. Folia Histochem. Cytochem. 10, 293 - 312 (1972).

Address' Dr. E. GREGORASZCZUK, Department of Ammal PhYSIOlogy, Institute of Zoology,

Jagielloman UniversIty, Krupmcza 50, PL . 30·060 Krakow.