Reprod. Dom. Anim., 28,273-278 (1993) 01993, Paul Parey Scientific Publishers, Berlin and Hamburg ISSN 0936-6768

Forschungsinstitut fur die Biologie landwirtschaftlicher Nutztiere, Dummerstod Germany

Effect of Prolactin Isolated from Human Amniotic Fluid on Cultured Bovine Cells of the Reproductive Tract and Splenocytes

U. Tiemann, B. Lohrke, J. Kowitz and H. Alm

Contents: Prolactin (PRL) was isolated from human amniotic fluid and than fractionated on Concanavalin A-Sepharose (ConA-S). Two fractions were tested for the 'H-thymidine incorporation in the DNA of bovine granulosa, oviductal and stromal cells as well as splenocytes. All cell .types reacted to PRL I or PRL I1 in a dose dependent manner. In general the effect of PRL I was greater than PRL II, and low concentrations of both enhanced the cell proliferation more than higher doses. The incorporating effect of 3H- thymidine under the influence of PRL I was higher in stromal cells and splenocytes than in granulosa and oviductal cells. PRL II did not have such a stimulating effect, even the presence of 14 ng/ml resulted in a decrease of 3H-thymidine incorporation in granulosa cells. The reason of this different intensity of tested PRL variants may depend on the molecular heterogeneity. The results indicate that PRL forms possess a cel type-specific effect on the proliferation of cells of the bovine female reproductive tract and splenocytes. Key words: prolactin; 3H-thymidine incorporation; splenocytes; granulosa, oviductal and uterine stromal cells; bovine

Inhalt: Wirkung von Prolaktin, isoliert aus humaner Amnionfliissigkeit, auf kultivierte Zellen des Reproduktionstraktes und der Milz vorn Rind Prolaktin (PRL) wurde aus humaner Amnionfliissigkeit isoliert und iiber Concanavalin A- Sepharose (ConA-S) fraktioniert. Zwei Formen wurden auf die 3H-Thymidin-Einbaurate in die DNA von Granulosa-, Eileiterepithel-, uterinen Stroma- und Miluellen beim Rind untersucht. Alle Zelltypen reagierten in ihrer Proliferation auf PRL I oder PRL II dosis- abhangig. Der Effekt von PRL I war im Vergleich zu PRL II groJer und beide Formen verursachten bei niedrigen Konzentrationen eine hohere proliferative Wirkung gegeniiber hoheren Dosen. Die 'H-Thymidin-Einbaurate war unter dem EinjluJ von PRL I bei Milz- und Stromazellen vermehrt gegeniiber Granulosa- und Eileiterepithelzellen. Wahrend- dessen PRL II nicht solch stimulierenden Effekt auf die Proliferation ausiibte und bei I4 ng/ml sogar eine Abnahme des 'H-Thymidineinbaus in Granulosazellen zu messen war. Die Ursache fiir diese unterschiedlichen Reaktionen konnte in der Heterogenitat der Prolaktinmolekiile begriindet sein. Die Ergebnisse zeigen, daJ PRL-Fonnen zelltyp-spezi- fisch auf die Proliferation von Zellen des weiblichen Reproduktionstraktes und der Milz beim Rind wirken.

Introduction

Numerous biological effects of prolactin (PRL) have been elucidated in mammals, including stimulating and inhibiting effects on the reproductive functions. Further PRL modulates the immune function in mice, rats and humans. T- and B-Lymphocytes possess PRL-receptors and can induce the proliferation on these cells (Russel et al., 1985, 1987; Biswas, 1992). The presence of specific receptors for PRL in human ovaries suggests that PRL directly regulates ovarian function (Saito and Saxena, 1975). Moreover, the hormone appears to play U.S. Clearance Center Statement: 0936-6768/93/2806-273 $02.50/0

214 U. Tiemann et a l

a role in coordination of ovarian activity, the state of the oviduct and of the uterus in preparing the implantation and maintenance of the pregnancy. Even in low concentration, PRL en- hanced the attachment of growth of human endometrial cells (Negami and Tominaga, 1991). Suppression of PRL expression abrogated the decidualization process - the prerequisite for a successful pregnancy (Kariya et al., 1992). Investigations of the biochemical nature of PRL have shown that this hormone exists in various molecular forms. Several studies have observed that the forms indicated different bioactivity (Lewis et al., 1988; Markoff et al., 1988; Subramanian et al., 1991; Andries et al., 1992). PRL has been shown to be heterogeneous when analyzed by gel filtration, chromatography or electrophoresis in serum or amniotic fluid (Fukuoka et al., 1991; Andries et al., 1992). The objective of the present study was to investigate the direct action of two PRL variants in reproductive and spleen cells. Thus the proliferation response to the PRL forms of granu- losa-, oviductal- and endometrial stromal cells as well as splenocytes was examined using the 3H-thymidine incorporation technique for the assessment of the bioactivity.

Material and Methods

Hormone: Prolactin was prepared from human amniotic fluid and characterized as described elsewhere (Lohrke et al., 1993). Briefly, the purified prolactin was fractionated by Concan- avalin A-Sepharose 4 B (ConA-S) and the PRL fractions were characterized by electrophoresis on 12,5% polyacrylamide gels, immunoblotting and glycan detection of the blotted proteins using commercial enzyme immunosorbent assays (ELISA) which are specialised to detect the nonglycosylated PRL form. The ConA-S affinity chromatography separated the forms in four fractions (I, 11, 111, IV). Fraction I contained nonglycosylated and glycosylated forms which distinguished from the other glycosylated forms by the glycan structure and thereby did not interact with the ConA-S. The retarded eluting Fraction 11 contained prolactins with scanty glycan amount (< 10 ng glycan moiety/pg prolactin protein). Both of these fractions (PRL I and PRL 11) were examined in the present study. Animals: Normally cycling heifers aged between 2 and 3 years were used for treatment and were slaughtered in the abattoir of the institute. Reproductive tracts and spleens with no abnormalities were removed from the carcasses immediately after slaughter.

Preparation of cells Granulosa cells: The ovaries were collected into Dulbecco’s PBS medium. Twenty minutes after slaughter granulosa cells were aspirated aseptically from follicles (2 to 4 mm in diameter) by means of a syringe and were flushed with Dulbecco’s PBS. To disperse cell clusters the cell aggregates were resuspended several times through a pasteur pipette. Subsequently the cells were given into RPMI-Medium. The cells were centrifugated at 200 x g for 5 min and resuspended in serum-free RPMI-medium. To determine the cell number, an aliquot of the cell suspension was counted in a hemocytometb. Cell viability was assessed by trypanblue exclusion. Oviductal cell: The oviducts were obtained from the local abattoir and were transported to the laboratory on ice within 20 minutes. In the laboratory the oviducts were strainghtened by trimming excess connective tissue, washed in TCM 199 (10% FCS) and dried. Oviducts were then flushed from the ovariadampullary end towards the utero tubal junction with 10 ml TCM 199 (10% FCS) into a test tube. The collected cell suspensions were centrifuged twice at 500 x g for 10 min. The pellet was resuspended in serum-free RPMI-medium and the cell number was counted. Stroma cells: Samples of bovine endometrium were aseptically removed from uteri and the stromal cells were obtained by the method described by Fortier et al. (1988). Briefly, pieces of endometrium were transferred into Hanks’ balanced salt solution (HBSS) without Ca2+ and Mg2+. After enzymatic removal of luminal epithelium, the tissue then was digested with Trypsin and Collagenase. The suspension containing stromal and glandular epithelium was subjected to gravity settling. The settled epithelium was removed and the supernatant

Effect of Prolactin on Cultured Bovine Cells 215

was centrifuged for 5 min at 1000 x g. The pelleted stromal cells were rinsed in HBSS, centrifuged, and resuspended in RPMI-Medium (10% FCS). The isolated stromal cells were grown as monolayers in 50 cm2 flasks and maintained in RPMI-medium for 5 days. The media were changed after 3 days. After the fifth day of incubation, cells were rinsed twice with HBSS, trypsinized and subcultured in serum-free RPm-medium. Splenocytes: The spleen was removed and placed into precooled PBS (pH7,4). A cell suspension was prepared by pressing the pieces of spleen through a plastic screen. The clumps were allowed to settle and then treated with 0,38% ammonium chloride to lyze erythrozytes. After this treatment the cells were washed twice in PBS and centrifuged at 500 x g for 5 min. Then the cells were resuspended into RPMI-medium (5% FCS). Cell culture: After cell counting and viability determination of cells, the cells were cultivated in 96 well tissue culture plates (Greiner, FRG). 2 x lo5 cells (granulosa-, oviductal- and stromal cells, splenocytes) 50 pl PRL I or II in following dilutions: 1,75; 3,5; 7,O and 14 ng/ ml were given and 50p1 RPMI-medium per well. Medium was substituted for PRL in control wells. After 48 h of incubation at 37 "C, in humified air, 5% CO,, 0,5 pCi 3H- thymidine (Nuclear Institute, Rossendorf-Berlin) were added to each well. The wells were harvested in glass fibre disks after 18 h using a semiautomated cell harvester (Skatron). Incorporation of 3H-thymidine into DNA of cells was measured in a scintillation counter.

Statistical analysis

The results are given by means k S.D. of three experiments in which the variants run in triplicate. Significance of differences between two means was tested by Student's t-test. Differences were compared between the control group and a test group in each case.

Results

To determine the bioactivity of the PRL forms in granulosa cells, the proliferation rate of the cells cultivated with and without the PRL forms were measured by aid of the 3H-thymidine incorporation. Figure 1 shows that PRL I caused a significant dose-dependent proliferative effect on granulosa cells. The highest stimulating effect (146,8%, P < 0,02) was obtained in 3,5 ng/ml. Contrasting PRL ZI exerted the highest stimulating effect on the thymidine

control 1,75 3,5

C C

7,O 14,O

ng prolactin/ml incubation volume

Fig. I : The 'H-thymidine incorporation by granulosa cells in vitro. Cells were incubated without (control) and with PRL I or PRL II in serumzfree RPMI-medium. Differences: P i 0,05 a and c,e; b and d - P < 0,02 a and b - P < 0.02 c and e

276 U. Tiemann et al

incorporation at 1,75 ng/ml (118%). Similar effects were obtained with the other two concentrations, whereover 14,O ng PRL IYml significantly decreases (69,9%, P < 0,05) the proliferation of granulosa cells in comparison with the controls. Between two PRL forms significant differences occur at 3,5 ng (P < 0,05) and 14 ng/ml (P < 0,02). Oviductal cells responsed to PRL I still more markedly than granulosa cells. Figure 2 demonstrates that even 1,75 ng/ml caused an increase in the 3H-thymidine incorporation of 164,5% (P < 0,Ol) and also the other concentrations significantly elevated the 3H-thymidine incorporation. However PRL II once stimulated the 3H-thyrnidine incorporation at 1,75 ng/ ml (155%, P < 0,Ol). The incorporation rates in the other concentrations were in the same range as the controls. Among the cell types from the reproductive organs, the PRL forms exerted the most striking proliferative effect on endometrial stromal cells (Fig. 3). The highest thymidine uptake into

25

20

15

10

5

C d cpm (x 1000)

control 1,75 3,5 7,O 14,O

ng prolactin/ml incubation volume - - EPRLI L P R L I I

Fig. 2: The 'H-thymidine uptake by cultured oviductal cells incubated without (control) and with PRL I or PRL 1I in serum-free RPMI-medium. DifSerences: P < 0,02 a and d - P < 0,Ol a and c

1200 -

control 1,75 3,5 7D 14.0

ng prolactin/ml incubation volume -- 7 L PRL I L ? PRL II

Fig. 3: The 'H-thymidine incorporation of cultured uterine stromal cells incubated without (control) and with PRL I or PRL I1 in serum-free RMPI-medium. Differences: P < 0,OS a and b; d and b - P < 0 ,Ol a and d; c and b - P < 0,001 a and c

Effect of Prolactin on Cultured Bovine Cells 211

the DNA of the cells was at 1,75 ng/ml of PRL I (247%, P < 0,001). The stimulating activity was dose-dependent, and even 14 ng/ml caused a stimulating effect of 199% (P < 0,Ol) compared to the control group. Also PRL 11 significantly increased the thymidine uptake in all concentrations, but these effects were lower than that of PRL I. Significant differences between the two forms occurred in the concentrations of 1,75; 7,O and 14,O ng/ml. Bovine splenocytes responded in the presence of PRL I with a dose-dependent increase of the 3H-thymidine incorporation (Figure 4). In a concentration of 1,75 ng/ml PRL I dramatically elevated the incorporation rate of spleen cells (375%; P < 0,001) compared to the controls. The stimulating effect decreased with increasing concentration (14 ng/ml) corresponded with 257% (P < 0,Ol) thymidine uptake in comparison with the control group. However, PRL 11 indicated only marked stimulating activity in the thymidine incorporation of the cells at low concentrations (1,75 and 3 3 ng/ml), whereas the incorporation rate was

d d

control 1,75 3.5 7.0 14,O

ng prolactin/ml incubation volume - - 2 3 PRL I ~ PRL I I

Fig 4 The 'H-thymidine incorporation of cultured splenocytes incubated without (control) and with PRL I or PRL II tn RPMI-medium (5% FCS) Differences P c 0,OI a and b, e, e and c - P c 0,001 a and d

not different from the control level at 14 ng/ml. Moreover, the effect of PRL I and 11 on the thymidine incorporation rate differed in all concentrations (P < 0,Ol).

Discussion

Little information is available concerning the relationship between PRL forms and the physiological function of such forms. Thus, we have attempted to assess the effects of two PRL fractions (PRL I, 11), which were characterized by their affinity to the lectin moiety of ConA-Sepharose and glycan moiety by glycan detection of the blotted prolactins as well as by immunotechniques (Lohrke et al., 1993). The PRL fraction I contained nonglycosylated forms and PRL with a glycan moiety which did not interact with ConA, whereas the prolactins of the fractionII were glycosylated to a small extent and the glycan moiety indicated low affinity to ConA conjugated to Sepharose 4B. Recently it has been accepted more and more that PRL acts on reproductive and immune levels. Therefore, the bioactivity of PRL I and 11 was examined in cultivated cells from bovine reproductive organs (ovarian granulosa, oviductal epithelial cells, endometrial stromal cells) and from the spleen using the 3H-thymidine incorporation rate as an indirect marker of the cell proliferation. From our experiment it is evident that PRL I and PRL 11 stimulated the proliferation of all cells; of course, the stimulation potency of PRL I was considerably higher than that of PRL 11. Thus, the PRL I forms appear to constitute the prolactin which was used in dose-response studies

27 8 U. Tiemann et al.

using human endometrial stromal cells (Negami et al., 1991), human luteal cells (Alia et al., 1987) or human and murine splenocytes (Hartmann et al., 1988; Biswas et al., 1992). These reports confirm our results concerning the dose-dependency of the mitogenic PRL I activity. However, the proliferative activity of PRL 11 from amniotic fluid has apparently not been studied yet. Bovine granulosa cells responded to a higher physiological dose (14 nglml) not only with abrogation but also with inhibition of the thymidine incorporation. The epithelial cells from the oviduct as well as the splenocytes abolished the proliferation response in PRL 11 presence (14 nglml) in contrast to the PRL I activity. This result may be consistent with findings recently reported (Subrarnanian et al., 1991) that the various PRL forms detectable in human follicular fluid exerted different effects on Nb2 cells, a rat node lymphoma cell line which is often used in testing the bioactivity of PRLI in vitro. Like the results presented here, the prolactins without affinity to ConA stimulated the lymphoma cell proliferation to a significantly higher extent than other forms. Thus, these reports and our findings tempt to speculate that a balance among the PRL forms may play a role in coordination of the reproductive and immune systems. PRL forms may coordinate the activation state in certain cells participating in reproductive function especially in signaling the follicular maturation to both the oviduct and the endometrium, which respond with preparing the reception of releasing oocytes or blastocystes, respectively.

References Aha, H.W., K.O. Rogo, and S. Gombe, 1987: Effects of prolactin and steroidogenesis by human luteal cells in

culture. Fertility and Sterility 47, 947-955. Andries, M., D. Tilemans, and C. Denef, 1992: Isolation of cleaved prolactin variants that stimulate DNA

synthesis in specific cell types in rat pituitary cell aggregates in culture. Biochem. J. 281, 393400. Biswas, R. and U. Chattopadhay, 1992: Altered prolactin response of the lymphocytes of tumor-haring mice.

Inter. J. Cancer 50, 93-100. Fortier, M.A., L A . Guibault, and F. Gross, 1988: Specific properties of epithel and strornd cells from the

endometrium of cows. J. Repro. Fertil. 83,239-248. Fukuoka, H., R. Hamaoto, and M. Higurashi, 1991: Heterogeneity of serum and amniotic fluid prolactin in

human. Horn. Res. 35 ,5843. Hartmann, D.A., E.W. Bernton, T.K. Shakarjian, and J.W. Holaday, 1988: Antibodies to prolactin inhibit

murine and human lymphocyte proliferation in vitro by inhibiting lymphocyte response to T- and B-cell growth factors. Feder. Amer. SOC. Exp. Biol. A 1642.

Kariya, M., H. Kanzaki, K. Takakura, K. Jmai, N. Okamoto, N. Emi, Y. Kariya, and T. Mori, 1992: Interleukin 1 inhibits in vitro decidualization of human endornetrial stromal cells. J. Clin. Endocrinol. Metabol. 73,

Lewis, U.JU., R.N.P. Sings, Y.N. Sinha and W.P. Vanderlaan, 1985: Glycosylated human prolactin. Endocrinology

Lohrke, B., B. Kriiger, and T. Viergutz, 1993: The glycosylation as source of the varibility in prolactin patterns

Markoff, E., D.W. Lee, and D.R. Hollingsworth, 1988: Glycosylated prolactin in serum during pregnancy. J.

Negami, A.I. and T. Tominaga, 1991: Effects of prolactin on cultured human endometrial cells. Horn. Res. 35

Russell, D.H., R. Kibler, L. Matrisian, D.F. Larson, G. Poulos, and B.E. Magun, 1985: Prolactin receptors on human T and B lymphocytes: antagonism of prolactin binding by cyclosporine. J. Immunol. 134,3027-3031.

Russell, D.H., A.R. Buckley, D.W. Montgomery, N.A. Larson, P.W. Gout, Ch.T. Beer, Ch.W. Putnam, Ch.F. Zukowski, and P.WW. Kibler, 1987: Prolactin-dependent mitogenesis in Nb 2 node lymphoma cells: effects of immunosuppressive cyclopeptide. J. Immunol. 138,276-284.

Saito, T. and B.B. Saxena, 1975: Specific receptors for prolactin in the ovary. Acta Endocrinol. (Copenhg.) 80,

Subramanian, M.G., A.G. Sacco, K.S. Moghissi, D.M. Lawson, andR.R. Gala, 1991: Prolactin size heterogeneity

1170-1 174.

116,359-365.

of individual human amniotic fluids. Biol. Chem. Hoppe-Seyler 374,271-279.

Clin. Endocrinol. Metabol. 67, 519-523.

(Suppl. 1) 5 c 5 7 .

126-137.

in human follicular fluid - a preliminary study. Inter. J. Fertil. 36, 367-371.

Submitted 8.10.1992

Address: Dr. rer.nat. Ute Tiemann, Forschungsinstitut fiir die Biologie landwirtschaftlicher Nutztiere, For- schungsbercich Fortpflanzungsbiologie, Wilhelm-Stahl-Allee 2, 18196 Dummerstorf, Germany.