Vol. 187, No. 3, 1992 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

September 30, 1992 Pages 1227-1231

REVERSE TRANSCRIPTION WITH NESTED POLVMERASE CHAIN REACTION SHOWS EXPRESSION OF BASIC FIBROBLAST GROWTH FACTOR TRANSCRH’TS

IN HUMAN GRANULOSA AND CUMULUS CELLS FROM IN VITRO FERTILISATION PATIENTS

Richard WATSON, Frederick ANTHONY, Mark PICKETI’*, Paul LAMBDEN*, Gordon M. MASSON and Eric J. THOMAS

Department of Obstetrics and Gynaecology, University of Southampton, Coxford Road, Princess Anne Hospital, Southampton, England SO9 4HA

*Department of Molecular Microbiology, South Laboratory and Pathology Block, Southampton General Hospital, Southampton, England SO9 4XY

Received August 6, 1992

Basic fibroblast growth factor (bFGF) is a potent angiogenic factor that has also been implicated in granulosa cell and oocyte maturation. We now report the expression of messenger ribonucleic acid (mRNA) encoding bFGF in human granulosa and cumulus cells obtained at oocyte recovery in in vitro fertiliiation patients. It was necessary to use the sensitive technique of a nested polymerase chain reaction (PCR) after reverse transcription (RT) to detect transcripts. This finding in conjunction with a recent report showing the presence of transcripts for transforming growth factor beta (TGFP) in the same type of cells by PCR indicates that mechanisms are in place for controlling extracellular proteolysis and cell differentiation. 0 1992 Academic Press. Inc.

Basic fibroblast growth factor (bFGF) is a 146 amino acid peptide that has been isolated from

a wide range of tissues (1). It has reported mitogenic and angiogenic activity and has been

implicated in the local modulation of ovarian function (2). The mRNA transcripts for bFGF

have been detected in rat ovaries but not without difficulties (2,3). The latter authors used

reverse transcriptase-polymerase chain reactions to detect the expression of bFGF mRNA in

dioestrus ovaries (2). However, there have been no reports until now on the detection of bFGF

mRNA expression in the human ovary. We endeavoured to detect bFGF transcripts in human

granulosa and cumulus cells taken from in vitro fertilisation (IVF) patients at the time of oocyte

recovery. LaPolt et al (4) suggested that bFGF acts as an intraovarian inducer of granulosa

cell tissue plasminogen activator (tPA) expression and oocyte maturation. The plasminogen

activator system is a general proteolytic system that is implicated in angiogenesis (5).

Increased production of plasmin or phospholipase C may be responsible for the release of

soluble complexes of bFGF bound to heparan sulphate proteoglycans (6). Phospholipase C

is produced in the periovulatory period in response to Luteinising Hormone (LH) for the release

0006-291X/92 $4.00

1227 Copyight 0 1992 by Academic Press, Inc.

All rights of reproduction in arq form reserved.

Vol. 187, No. 3, 1992 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of arachidonic acid for prostaglandin production. The activity of a negative regulator of

angiogenesis, transforming growth factor beta (TGFP), is also regulated by proteases and

activated by plasminogen activator and plasmin (5). TGFPl and TGFP2 mRNA have very

recently been detected in human granulosa-luteal and cumulus cells (7). We now report, using

a more sensitive technique, the expression of bFGF in human granulosa cells from a similar

group of patients to those previously studies (5).

MATERIALS AND METHODS

Patients. Women attending the Southampton in vitro fertilisation (IVF) programme were superovulated as previously described (8) with human menopausal gonadotrophin (hMG, Serono, Welwyn Garden City, England) from day four of menses after down-regulation of the anterior pituitary with a luteinising hormone releasing hormone agonist, buserelin (Hoechst, London, England). When the leading follicles were > 18mm diameter, human chorionic gonadotrophin (hCG) was administered intramuscularly and 34 hours later follicles were aspirated for oocyte recovery.

Cumulus and granulosa cell preparations. Cumulus cell clumps, free of oocytes, identified microscopically in culture medium one hour after transvaginal ultrasound-guided follicle aspiration, were drawn up into pipettes and then dispersed in RNAzol (Product Code No. 8050- 4004, Biogenesis Ltd., Bournemouth, England). RNAzol is a guanidinium thiocyanate-phenol mixture developed for optimal isolation of total RNA. The manufacturers recommendations were followed for RNA extraction and isopropanol precipitation before reconstitution in nuclease free water. Reverse transcription was carried out using Stratagene first strand kit (Catalogue No. 200420, Stratagene, California, United States) and random primers. First round PCR was run for 30 cycles with cDNA from < 0. lpg RNA, 2.5 U Thermus aquaticus (Taq) polymerase (Promega, Southampton, England) and primer set 5’ to 3’ of sense- CCAAGCGGCTGTACTGCAA and antisense- CAGCTCTTAGCAGACATTGG at 72,91 and 58 degrees Celsius for a 382 base pair product (9). One ~1 of a total 100~1 first round product was used in the second round of 30 cycles with a primer set of sense- TCTTCCTGCGCATCCACC and antisense- CAATGCCACATACCAACTG at 72, 91 and 56 degrees Celsius to generate a 266 base pair product,

Granulosa cells were also prepared from follicle washes after initial follicular aspiration. These cells were purified by density gradient centrifugation on Percoll (Product code 17-0891- 01, Pharmacia LKB, Milton Keynes, England) and identified microscopically before RNAzol extraction and procedures as above.

Analysis of RT-PCR products was carried out by electrophoresis on 1.5% agarose gels and also by Taq cycle sequencing. Amplified DNA was purified by Magic PCR Minipreps (Cat. No. A7090, Promega, Southampton, England) before sequencing. Taq cycle sequencing was run for 25 cycles with one of the primers, free nucleotides and fluorescent dideoxy terminators. Polyacrylamide gel electrophoresis was scanned in an Applied Biosystems Model 373A computer linked automatic sequencing system.

RESULTS

Cumulus cell clumps, two preparations, and granulosa cell preparations, three, all yielded an

RT-nested PCR product of 266 base pairs. This product was compared with that produced by

an erythro-leukemia cell line (K562) known to produce bFGF. In addition a reagent extraction

1228

Vol. 187, No. 3, 1992 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Fig. 1. Nested PCR of cDNA produced from human bFGF mRNA. Photograph shows agarose gel electrophoresis of ladder (Gibco BRL, Paisley, Scotland) and PCR products:- from left to right lane 1) 100 base pair ladder 2) PCR run with outer primers and cDNA of erythroleukemia cell line (control) 3) Negative extraction with outer primers 4) Granulosa cell cDNA with outer primers 5) Enzyme, outer primers, nucleotides and no template 6) Positive control with inner primers 7) Negative extraction with inner primers 8) Granulosa cell first PCR product with inner primers 9) Reagent negative control with inner primers 10) 100 base pair ladder.

negative control and PCR reagents without template negative control were prepared and

compared with the above. Figure 1 shows the comparison of a granulosa cell preparation with

that of K562 and the two negative controls. Product is not visualised with the outer primers

designed to generate the 382 base pair product in lanes 2-5, but the 266 base pair product

generated from the inner primers is visible for the K562 and granulosa cell preparation in lanes

7 and 9 respectively. The purified PCR DNA from the granulosa cell preparation gave the

following sense sequence with primer TCTTCCTGCGCATCCACC and Taq cycle sequencing:

“TC TTC CTG CGC ATC CAC C”CC GAC GGC CGA GTT GAC GGG GTC CGGGAGAAGAGCGACCCTCACATCAAGCTACAACTTCAAGCA GAA GAG AGA GGA GTT GTG TCT ATC AAA GGA GTG TGT GCT AAC CGTTACCTGGCTATGAAGGAAGATGGAAGATTACTGGCTTCT AAA TGT GTT ACG GAT GAG TGT TX TIT ‘I-l-l- GAA CGA TTG GAA TCT AAT AAC TAC AAT ACT TAC CGG TCA AGG AAA TAC AC”C AGT TGG TAT GTG GCA TTG”

1229

Vol. 187, No. 3, 1992 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

- Sequenced 266 bp product with sense version of primers for nested reaction in inverted

commas.

This sequence agrees with that reported in the literature for the bFGF mRNA transcript and

would encompass 2 introns in the genome (10).

DISCUSSION

The detection of transcripts for bFGF in human granulosa and cumulus cells provides us with

another important piece of information on the angiogenesis-extracellular proteolysis mechanisms

in this particular cell system. The growth factor bFGF is another controlling factor necessary

for the transformation of granulosa cells to luteal cells along with TGFPI and TGFP2. As has

been already suggested bFGF is probably present to effect tPA expression (4, 6). This would

initially be brought about by bFGF produced in granulosa or cumulus cells being released in a

heparan sulphate proteoglycans complex from cell surfaces by phospholipase C (4, 6). The

phospholipase C itself would have been produced by the LH surge in the periovulatory period

or the exogenous hCG administered in the IVF cycle that mimics LH. Whether the expression

of bFGF transcripts occurs after, during or before this event is open to further study. Certainly

TGFP will be playing a part in this elaborate mechanism and will be modulating bFGF action.

The released latent form of TGFP is activated by plasminogen activators and plasmin and the

TGFP then induces production of an inhibitor of plasminogen activator (5). The present report

and that of Mulheron and colleagues (7) suggest that the mechanisms for controlling

extracellular proteolysis are in place in human granulosa cells.

1.

2.

3.

4.

5.

6.

REFERENCES

Gospodarowicz, D., Neufeld, G. and Schweiger, L. (1986) Cell. Diffn. 19, 1-17.

Hearn, M.T.W. (1991) Clin. End. Metab. 5(4), 571-593.

Guthridge, M., Schmitt, J., Bertolini, J., Cowling, J., Runting, A., Katsahambas, S., Drummond, A.E. and Hearn,M.T.W. (1992) In: Angiogenesis, edited by Steiner, R., Weisz, P.B. and Langer, R. p.219-229, Birkhauser Verlag, Basel.

LaPolt,P.S., Yamoto,M., Veljkovic,M., Sincich, C., Ny T., Tsafriri, A. and Hsueh, A.J.W. (1990) Endocrinology 127(5), 2357-2363.

Rifkin, D.B., Moscatelli, D., Bizik, J., Quarto, N. Blei, F., Dennis, P., Flaumenhaft, R. and Mignatti, P. (1990) Cell. Differ. Dev. 32(3), 313-318.

Brunner, G., Gabrilove, J., Rifkin, D.B. and Wilson, E.L. (1991) J. Cell. Biol. 114(6), 1275-1283.

1230

Vol. 187, No. 3, 1992 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

7. Mulheron, G.W., Bossert, N.L. Lapp, J.A., Walmer, D.K. and Schomberg, D.W. (1992) J. Clin. End. Metab. 74(2), 458-460.

8. Smith, E.M., Anthony, F. Gadd, S.C. and Masson, G.M. (1989) Br. Med. J. 298, 1483-1486.

9. Innis, M.A., Gelfand, D.H., Sninsky, J.J. and White, T.J. (1990) PCR Protocols. Academic Press, California.

10. Abraham, J.A., Whang, J.L. Tumulo, A. Mergia, A., Friedman, J., Gospodarowicz, D. and Fiddes, J.C. (1986) EMBO J. 5(10), 2523-2528.

1231