J. Endocrinol. Invest. 13: 301-305, 1990
Immunohistochemicallocalization of growth hormone-releasing hormone in human gonads C. Moretti*, A. Fabbri**, L. Gnessi*, V. Bonifacio*, M. Bolotti, M. Arizzi*, Q. Nazzicone***, and G. Spera*
*Clinica Medica v, Universita degli Studi di Roma "La Sapienza", Policlinico Umberto I, **Cattedra di Andrologia, Universita degli Studi di Roma "La Sapienza" and ***Divisione I Ostetricia e Ginecologia, Ospedale San Camillo, Roma ABSTRACT. Human gonads were examined for the presenee of ir growth hormone-releasing-hormone. We demonstrated the presenee of immunostainable eells both in ovarian and in testieular tissue using a non-erossreaetive antiGRH antiserum and the immunoperoxidase deteetion teehnique. In ovaries from ovulating women, GRH immunoreaetivity was loealized in the eorporea lutea; granulosa eells, theea eells and eells of primary
follieles did not stain. In premenopausal ovaries, staining was deteetable in seattered luteinized stromal eells. In testes from post-puberal men GRH immunoreaetivity was loealized in the Leyding eells; eells of the germinal epithelium did not stain. These results demonstrate the presenee of GRH in human gonads and suggest that this peptide may exert regulatory funetion at the testieular and ovarian levels.
INTRODUCTION A large number of peptides have been found to be produced locally within the gonads, and seem to play both paracrine and autocrine roles as mediators of communication among cells (1,3). Recently, high concentrations of immunoreactive CRF (irCRF) were reported in the testis (4), which also contains other hypothalamic neuropeptides including TRH, GnRH and POMC-derived peptides (2, 3, 5-7). Like other neuropeptides originally described as primarily of hypothalamic origin, the 44 AA peptide GRH has also been found in peripheral tissues including several segments of the gastrointestinal tract (8, 9), human placenta (10) and pancreas (11). Recently a rat GRH-like substance and its messenger RNA has been identified in rat testis (12). We investigated the presence and distribution of ir-GRH in specimens from normal human testes and ovaries, in order to determine whether this peptide has potential paracrine actions in the gonads.
MATERIALS AND METHODS Ovarian tissue was obtained from oophorectomy specimens (for non-ovarian causes) from normal menstruating women and from premenopausal patients with clinical manifestations of a luteal defect. Testicular tissue was obtained from routine biopsies performed in infertile patients. Specimens were fixed in Bouin's solution, embedded in paraffin wax and sectioned at 5-7 microns. For the immunocytochemical visualization of GRH, the peroxidase-antiperoxidase (PAP) technique was performed by using a commercial kit (Ortho Diagnostic System, Raritan, N.J.). The antiserum was used at a diluition of 1:1 00 and 1:300. Anti-human GRH (1-44)-NH 2 antiserum was supplied by Peptide (Scientific marketing associates, London England). The cross-reactivity of the antiserum was 100% with porcine GRH, 30% with human GRH (30-44)NH 2, < 0,1 % with human GRH (1-29). No cross-reaction against human CRF, VIP, human NPY, human beta-endorphin and somatostatin was observed. Control slices were incubated in phosphate-buffered saline (PBS). Preabsorbtion of the antiserum (100 pg/ml diluted antiserum) with excess of synthetic human GRH (1-44)-NH 2 (Amersham, USA) was also performed.
Key-words: GRH. testls, ovary Correspondence: Costanzo Moretti, MD., Clinlca Medica V Unlversitil di Roma "La Sap,enza". Policlmico Umberto I, 00161 Rome, Italy.
Received Apnl 3, 1989; accepted January 31, 1990.
301
C. Moretti, A. Fabbri, L. Gnessi, et al.
Fig. 1 - (A): Immunohistochemical staining of GHRH in human testis. The graup of Leydig cells are strangly positive (original magnification x 500). (8): Portion of the same slice of human testis (contral) immunostained using preabsorbed antibody; staining was completely abolished (original magnification x 500).
RESULTS The indirect immunoperoxidase technique with antiGRH antiserum localized GRH immunoreactivity in tissue sections of both testis and ovaries. In the testis the irGRH was localized in groups of Leydig cells, which showed finely granular cytoplasmatic staining; negative immunoreactivity was observed in the tubule wall (Fig. 1A). No positive immunostaining was observed in the contral sections (Fig. 1B). In the ovarian tissue, detectable irGRH was observed only in scattered luteinized cells. In ovulating and premenopausal normalovaries, strang specific staining was localized in scattered theca derivedcells of corpora lutea (small luteal cells) (Fig. 2A). No positive immunostaining was observed in the contra I section (Fig. 2B). This staining was in the form of more sparse cytoplasmic granules than in Leydig cells. Granulosa cells and theca cells of follicles in different stages
of maturation were completely negative. No immunostaining was detected on control slices after incubation with PBS, preadsorbed with excess of GRH or normal serum, indicating that the staining was specific. Both in testis and in ova ries the immunopositivity corresponded to the same cell morphotype in all the sections prepared and screened. Three experiments were performed with a mean of 15 sections examined each. DISCUSSION These studies have demonstrated the presence of GRH immunoreactivity in the interstitial cellular compartment of human testis and ovary. Recently, Berry and Pescovitz (12) showed that GRH-like material is present in the testis and is locally synthesized, indicating a testicular origin of this peptide hormone. The amount detected from these Authors in the rat testis and hypothalamus were respectively of 1.62 ± 0.17 ng/g tissue and 2.7 ± 0.24 g/tissue.
302
GRH in human gonads
Fig. 2 - (A): Ovarian stroma with scattered theca-derived small cells (type I), localized in the interstitial tissue, immunopositive tor GHRH (original magnitication x 100). (8): Portion ot the same slice of human ovaries (control) immunostained using preabsorbed antibody; staining was completely abolished (original magnification x 500).
scattered luteal cells. The cells of the corpus luteum appear to arise from at least two different types of follicular steroid-secreting cell, theca-derived - (type I) and granulosa derived - (type II cells), that are morphc:ilogically distinct. Our immunopositive cells showed a diameter of about 20 /lm, and were spindle-shaped with an irregularly shaped nucleus, and therefore belonged to the theca lutein type I cells. No immunopositivity was observed in the larger granulosa-derived type 11 cells. We have recently observed that GRH is able to amplify the in vivo effect of FSH on follicular selecti on and growth (20). We showed that the intravenous administation of GRH from the 1st to the 7th day of cycle may induce follicular growth in women resistent to gonadotropin therapy who lack folliculogenesis and determine ovulation when associated with FSH therapy. Thus the actions of GRH in the ovary could be related to the initiation of growth of primordial follicles and to the differentiation of granulosa cells. This effect is probably related to a GH-dependent increase of interstitiallevels of IGF-
Our observation of immunohistochemical localization of irGRH in Leydig cells suggests that these cells could be the site of synthesis in the human testis. These findings are in line with the immunohistochemical identification of many other polypeptides that exert a messenger and proliferative functi on in the human testis: for example, insulin-like growth tactor (13), beta-endorphin (14), substance P (15) and bombesin (16). The presence of these neuropeptides in Leydig cells is intriguing and raises the question about the possible embryonic origin ot these cells trom the neural crest, as has been suggested by others (15). Also, IGF-I and ir-beta-endorphin have been detected, like ir-GRH, in human semen (14, 15). Beta-endorphin was also identified in testicular specimens of human hyperplastic interstitial tissue (18). The presence of a large number of hypothalamic peptides in the ovary has also been demonstrated (19), possibly related to the autocrine and/or paracrine regulation of gonadal function and development. We have localized GRH immunoreactivity in
303
C. Moretti, A. Fabbri, L. Gnessi, et al.
C production: extra-hypothalamic localization and possible functional significance. Acta Endocrinol. (Copenh.)276 (Suppl): 34, 1986.
I; however the present demonstration of the presence of irGRH within the human ovary may suggest a direct local action of the peptide. Furthermore, the immunohistochemical localization of the peptide in theca-derived luteal cells indicates a possible role of the peptide in formation and regulation of corpus luteum. The finding of GRH in human gonads is novel and confirms recent observations in animal studies. Further investigations will be necessary to clarify whether this peptide has a role in regulating gonadal function at the peripherallevel.
9. Bruhn 1.0., Mason R.T., Vale WW. Presence of growth hormone-releasing factor-like immunoreactivity in rat duodenum. Endocrinology 117: 1710, 1985. 10. Baird A, Wehrenberg W.B., Bohlen P., Ling N. Immunoreactive and biologically active growth hormone-releasing factor in the rat placenta. Endocrinology 117: 1598, 1985. 11. Shibasaki 1., Kiyosawa Y., Masuda A, Nakahara M., Imaki 1., Wakabayashi 1., Demura H., Shizume K., Ling N. Distribution of growth hormone-releasing hormonelike immunoreactivity in human tissue extracts. J. Clin. Endocrinol. Metab.59: 263, 1984.
REFERENCES 1. Sharpe R.M. Intragonadal factors controlling testicular function. Biol. Reprod. 30: 29, 1984.
12. Berry SA, Pescovitz O.H. Identification of a rat GHRH-like substance and its messenger RNA in rat testis. Endocrinology 123: 661, 1988.
2. Bardin C.w., Shaha C., Mather J., Salomon Y., Krieger D.T. Identification and possible function of pro-opiomelanocortin-induced peptides in the testis. Ann. NY Acad. Sci. 438: 346, 1984.
13. Vannelli B.G., Natali A, Barni 1., Serio M., Orlando C., Balboni G.C. Insulin-like growth factor-I (IGF-I) and IGF-I receptor in human testis: an immunohistochemical study. Fertil. Steril. 49: 666, 1988.
3. Fabbri A, Knox G., Buczlo E., Dufau M.L. Beta-endorphin production by the fetal Leydig cell: regulation and implications for paracrine control of Sertoli cell function. Endocrinology 122: 749, 1988.
14. Fraioli F., Fabbri A, Gnessi L., Silvestroni L., Moretti C., Redi F., Isidori A Beta-endorphin, met-enkephalin and calcitonin in human semen: evidence for a possible role in human sperm motility. Ann. NY Acad. Sci. 438: 364, 1983.
4. Yoon D.J., Sklar C., David R. Presence of immunoreactive corticotropin-releasing factor in rat testis. Endocrinology 122: 759, 1988.
15. Schulze W., Davidoff M.S., Holstein AF. Are Leydig cells of neural origin? Substance P-like immunoreactivity in human testicular tissue. Acta Endocrinol. (Copenh) 115: 337, 1987.
5. Morley J.M., Meyer N... Pekary AE., Melmed S., Carlton H.E., Briggs J.E., Hershman J.M. A prolactin inhibitory factor with immunocharacteristics similar to thyrotropin re leasing fador (TRH) is present in rat pituitary tumors (GH3 and W5), testicular tissue and a plant material, alfalfa. Biochem. Biophys. Res. Comm. 96: 47,1980.
16. Gnessi L., Isidori A, Bolotti M., Altamura S., Ulisse S., Jannini E.A, Fabbri A, Spera G. Identification of immunoreactive gastrin-releasing peptide related substances in adult Leydig cells. Endocrinology 124: 558, 1989.
6. Bhasin S., Heber D., Peterson M., Swerdloff R. Partial isolation and characterization of testicular GnRH-like factors. Endocrinology 112: 1144, 1983.
17. Baxter R.C., Martin J.L., Handelsman D.J. Identification of human semen insulin-like growth factor I/somatomedin-C immunoreactivity and binding protein. Acta Endocrinol. (Copenh) 106: 420,1984.
7. Pintar J.E., Schachter B.S., Herman AB., Durgerian S., Krieger D.T. Characterization and localization of proopiomelanocortin messenger RNA in the adult rat testis. Science 225: 632, 1984.
18. Spera G., Di Benedetto E., Falaschi P., Fabbri A, Lauro G., Bolotti M. Immunohistochemicallocalization of Beta-endorphin in hyperplastic interstitial tissue of the human testis. J. Androl. 9: 78, 1988. 19. Hsueh AJW., Adashi E.Y., Jones P.B.C., Welsh T.H. jr.
8. Thorner M.O., Vance M.L., Evans W.S., Ho K., Rogol AD., Blizzard R.M., Furlanetto R., Rivier L., Vale W. Growth hormone-releasing factor and somatomedin-
304
GRH in human gonads
induced ovarian folliculogenesis in women with anovulatory infertility. Reproductive Medicine: Medical Therapy In: G. Frajese, E. Steinberg, L.J. Rodriguez-Rigau (Eds.), Proceedings of the Second International Symposium on Reproductive Medicine, Fiuggi, Italy. Elsevier Science Publishers 875: 103, 1989.
Hormonal regulation of the differentiation of cultured ovarian granulosa cells. Endocr. Rev. 5: 76, 1984. 20. Moretti C., Fabbri A., Gnessi L., Forni L., Fraioli F., Frajese G. GHRH stimulates follicular growth and amplifies FSH-
305
Immunohistochemicallocalization of growth hormone-releasing hormone in human gonads C. Moretti*, A. Fabbri**, L. Gnessi*, V. Bonifacio*, M. Bolotti, M. Arizzi*, Q. Nazzicone***, and G. Spera*
*Clinica Medica v, Universita degli Studi di Roma "La Sapienza", Policlinico Umberto I, **Cattedra di Andrologia, Universita degli Studi di Roma "La Sapienza" and ***Divisione I Ostetricia e Ginecologia, Ospedale San Camillo, Roma ABSTRACT. Human gonads were examined for the presenee of ir growth hormone-releasing-hormone. We demonstrated the presenee of immunostainable eells both in ovarian and in testieular tissue using a non-erossreaetive antiGRH antiserum and the immunoperoxidase deteetion teehnique. In ovaries from ovulating women, GRH immunoreaetivity was loealized in the eorporea lutea; granulosa eells, theea eells and eells of primary
follieles did not stain. In premenopausal ovaries, staining was deteetable in seattered luteinized stromal eells. In testes from post-puberal men GRH immunoreaetivity was loealized in the Leyding eells; eells of the germinal epithelium did not stain. These results demonstrate the presenee of GRH in human gonads and suggest that this peptide may exert regulatory funetion at the testieular and ovarian levels.
INTRODUCTION A large number of peptides have been found to be produced locally within the gonads, and seem to play both paracrine and autocrine roles as mediators of communication among cells (1,3). Recently, high concentrations of immunoreactive CRF (irCRF) were reported in the testis (4), which also contains other hypothalamic neuropeptides including TRH, GnRH and POMC-derived peptides (2, 3, 5-7). Like other neuropeptides originally described as primarily of hypothalamic origin, the 44 AA peptide GRH has also been found in peripheral tissues including several segments of the gastrointestinal tract (8, 9), human placenta (10) and pancreas (11). Recently a rat GRH-like substance and its messenger RNA has been identified in rat testis (12). We investigated the presence and distribution of ir-GRH in specimens from normal human testes and ovaries, in order to determine whether this peptide has potential paracrine actions in the gonads.
MATERIALS AND METHODS Ovarian tissue was obtained from oophorectomy specimens (for non-ovarian causes) from normal menstruating women and from premenopausal patients with clinical manifestations of a luteal defect. Testicular tissue was obtained from routine biopsies performed in infertile patients. Specimens were fixed in Bouin's solution, embedded in paraffin wax and sectioned at 5-7 microns. For the immunocytochemical visualization of GRH, the peroxidase-antiperoxidase (PAP) technique was performed by using a commercial kit (Ortho Diagnostic System, Raritan, N.J.). The antiserum was used at a diluition of 1:1 00 and 1:300. Anti-human GRH (1-44)-NH 2 antiserum was supplied by Peptide (Scientific marketing associates, London England). The cross-reactivity of the antiserum was 100% with porcine GRH, 30% with human GRH (30-44)NH 2, < 0,1 % with human GRH (1-29). No cross-reaction against human CRF, VIP, human NPY, human beta-endorphin and somatostatin was observed. Control slices were incubated in phosphate-buffered saline (PBS). Preabsorbtion of the antiserum (100 pg/ml diluted antiserum) with excess of synthetic human GRH (1-44)-NH 2 (Amersham, USA) was also performed.
Key-words: GRH. testls, ovary Correspondence: Costanzo Moretti, MD., Clinlca Medica V Unlversitil di Roma "La Sap,enza". Policlmico Umberto I, 00161 Rome, Italy.
Received Apnl 3, 1989; accepted January 31, 1990.
301
C. Moretti, A. Fabbri, L. Gnessi, et al.
Fig. 1 - (A): Immunohistochemical staining of GHRH in human testis. The graup of Leydig cells are strangly positive (original magnification x 500). (8): Portion of the same slice of human testis (contral) immunostained using preabsorbed antibody; staining was completely abolished (original magnification x 500).
RESULTS The indirect immunoperoxidase technique with antiGRH antiserum localized GRH immunoreactivity in tissue sections of both testis and ovaries. In the testis the irGRH was localized in groups of Leydig cells, which showed finely granular cytoplasmatic staining; negative immunoreactivity was observed in the tubule wall (Fig. 1A). No positive immunostaining was observed in the contral sections (Fig. 1B). In the ovarian tissue, detectable irGRH was observed only in scattered luteinized cells. In ovulating and premenopausal normalovaries, strang specific staining was localized in scattered theca derivedcells of corpora lutea (small luteal cells) (Fig. 2A). No positive immunostaining was observed in the contra I section (Fig. 2B). This staining was in the form of more sparse cytoplasmic granules than in Leydig cells. Granulosa cells and theca cells of follicles in different stages
of maturation were completely negative. No immunostaining was detected on control slices after incubation with PBS, preadsorbed with excess of GRH or normal serum, indicating that the staining was specific. Both in testis and in ova ries the immunopositivity corresponded to the same cell morphotype in all the sections prepared and screened. Three experiments were performed with a mean of 15 sections examined each. DISCUSSION These studies have demonstrated the presence of GRH immunoreactivity in the interstitial cellular compartment of human testis and ovary. Recently, Berry and Pescovitz (12) showed that GRH-like material is present in the testis and is locally synthesized, indicating a testicular origin of this peptide hormone. The amount detected from these Authors in the rat testis and hypothalamus were respectively of 1.62 ± 0.17 ng/g tissue and 2.7 ± 0.24 g/tissue.
302
GRH in human gonads
Fig. 2 - (A): Ovarian stroma with scattered theca-derived small cells (type I), localized in the interstitial tissue, immunopositive tor GHRH (original magnitication x 100). (8): Portion ot the same slice of human ovaries (control) immunostained using preabsorbed antibody; staining was completely abolished (original magnification x 500).
scattered luteal cells. The cells of the corpus luteum appear to arise from at least two different types of follicular steroid-secreting cell, theca-derived - (type I) and granulosa derived - (type II cells), that are morphc:ilogically distinct. Our immunopositive cells showed a diameter of about 20 /lm, and were spindle-shaped with an irregularly shaped nucleus, and therefore belonged to the theca lutein type I cells. No immunopositivity was observed in the larger granulosa-derived type 11 cells. We have recently observed that GRH is able to amplify the in vivo effect of FSH on follicular selecti on and growth (20). We showed that the intravenous administation of GRH from the 1st to the 7th day of cycle may induce follicular growth in women resistent to gonadotropin therapy who lack folliculogenesis and determine ovulation when associated with FSH therapy. Thus the actions of GRH in the ovary could be related to the initiation of growth of primordial follicles and to the differentiation of granulosa cells. This effect is probably related to a GH-dependent increase of interstitiallevels of IGF-
Our observation of immunohistochemical localization of irGRH in Leydig cells suggests that these cells could be the site of synthesis in the human testis. These findings are in line with the immunohistochemical identification of many other polypeptides that exert a messenger and proliferative functi on in the human testis: for example, insulin-like growth tactor (13), beta-endorphin (14), substance P (15) and bombesin (16). The presence of these neuropeptides in Leydig cells is intriguing and raises the question about the possible embryonic origin ot these cells trom the neural crest, as has been suggested by others (15). Also, IGF-I and ir-beta-endorphin have been detected, like ir-GRH, in human semen (14, 15). Beta-endorphin was also identified in testicular specimens of human hyperplastic interstitial tissue (18). The presence of a large number of hypothalamic peptides in the ovary has also been demonstrated (19), possibly related to the autocrine and/or paracrine regulation of gonadal function and development. We have localized GRH immunoreactivity in
303
C. Moretti, A. Fabbri, L. Gnessi, et al.
C production: extra-hypothalamic localization and possible functional significance. Acta Endocrinol. (Copenh.)276 (Suppl): 34, 1986.
I; however the present demonstration of the presence of irGRH within the human ovary may suggest a direct local action of the peptide. Furthermore, the immunohistochemical localization of the peptide in theca-derived luteal cells indicates a possible role of the peptide in formation and regulation of corpus luteum. The finding of GRH in human gonads is novel and confirms recent observations in animal studies. Further investigations will be necessary to clarify whether this peptide has a role in regulating gonadal function at the peripherallevel.
9. Bruhn 1.0., Mason R.T., Vale WW. Presence of growth hormone-releasing factor-like immunoreactivity in rat duodenum. Endocrinology 117: 1710, 1985. 10. Baird A, Wehrenberg W.B., Bohlen P., Ling N. Immunoreactive and biologically active growth hormone-releasing factor in the rat placenta. Endocrinology 117: 1598, 1985. 11. Shibasaki 1., Kiyosawa Y., Masuda A, Nakahara M., Imaki 1., Wakabayashi 1., Demura H., Shizume K., Ling N. Distribution of growth hormone-releasing hormonelike immunoreactivity in human tissue extracts. J. Clin. Endocrinol. Metab.59: 263, 1984.
REFERENCES 1. Sharpe R.M. Intragonadal factors controlling testicular function. Biol. Reprod. 30: 29, 1984.
12. Berry SA, Pescovitz O.H. Identification of a rat GHRH-like substance and its messenger RNA in rat testis. Endocrinology 123: 661, 1988.
2. Bardin C.w., Shaha C., Mather J., Salomon Y., Krieger D.T. Identification and possible function of pro-opiomelanocortin-induced peptides in the testis. Ann. NY Acad. Sci. 438: 346, 1984.
13. Vannelli B.G., Natali A, Barni 1., Serio M., Orlando C., Balboni G.C. Insulin-like growth factor-I (IGF-I) and IGF-I receptor in human testis: an immunohistochemical study. Fertil. Steril. 49: 666, 1988.
3. Fabbri A, Knox G., Buczlo E., Dufau M.L. Beta-endorphin production by the fetal Leydig cell: regulation and implications for paracrine control of Sertoli cell function. Endocrinology 122: 749, 1988.
14. Fraioli F., Fabbri A, Gnessi L., Silvestroni L., Moretti C., Redi F., Isidori A Beta-endorphin, met-enkephalin and calcitonin in human semen: evidence for a possible role in human sperm motility. Ann. NY Acad. Sci. 438: 364, 1983.
4. Yoon D.J., Sklar C., David R. Presence of immunoreactive corticotropin-releasing factor in rat testis. Endocrinology 122: 759, 1988.
15. Schulze W., Davidoff M.S., Holstein AF. Are Leydig cells of neural origin? Substance P-like immunoreactivity in human testicular tissue. Acta Endocrinol. (Copenh) 115: 337, 1987.
5. Morley J.M., Meyer N... Pekary AE., Melmed S., Carlton H.E., Briggs J.E., Hershman J.M. A prolactin inhibitory factor with immunocharacteristics similar to thyrotropin re leasing fador (TRH) is present in rat pituitary tumors (GH3 and W5), testicular tissue and a plant material, alfalfa. Biochem. Biophys. Res. Comm. 96: 47,1980.
16. Gnessi L., Isidori A, Bolotti M., Altamura S., Ulisse S., Jannini E.A, Fabbri A, Spera G. Identification of immunoreactive gastrin-releasing peptide related substances in adult Leydig cells. Endocrinology 124: 558, 1989.
6. Bhasin S., Heber D., Peterson M., Swerdloff R. Partial isolation and characterization of testicular GnRH-like factors. Endocrinology 112: 1144, 1983.
17. Baxter R.C., Martin J.L., Handelsman D.J. Identification of human semen insulin-like growth factor I/somatomedin-C immunoreactivity and binding protein. Acta Endocrinol. (Copenh) 106: 420,1984.
7. Pintar J.E., Schachter B.S., Herman AB., Durgerian S., Krieger D.T. Characterization and localization of proopiomelanocortin messenger RNA in the adult rat testis. Science 225: 632, 1984.
18. Spera G., Di Benedetto E., Falaschi P., Fabbri A, Lauro G., Bolotti M. Immunohistochemicallocalization of Beta-endorphin in hyperplastic interstitial tissue of the human testis. J. Androl. 9: 78, 1988. 19. Hsueh AJW., Adashi E.Y., Jones P.B.C., Welsh T.H. jr.
8. Thorner M.O., Vance M.L., Evans W.S., Ho K., Rogol AD., Blizzard R.M., Furlanetto R., Rivier L., Vale W. Growth hormone-releasing factor and somatomedin-
304
GRH in human gonads
induced ovarian folliculogenesis in women with anovulatory infertility. Reproductive Medicine: Medical Therapy In: G. Frajese, E. Steinberg, L.J. Rodriguez-Rigau (Eds.), Proceedings of the Second International Symposium on Reproductive Medicine, Fiuggi, Italy. Elsevier Science Publishers 875: 103, 1989.
Hormonal regulation of the differentiation of cultured ovarian granulosa cells. Endocr. Rev. 5: 76, 1984. 20. Moretti C., Fabbri A., Gnessi L., Forni L., Fraioli F., Frajese G. GHRH stimulates follicular growth and amplifies FSH-
305
