Transferrin and somatomedin C receptors in the human ovarian follicles

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1 FERTILITY AND STERILITY Copyright c 1987 The American Fertility Society Printed in U.S.A. Transferrin and somatomedin C receptors in the human ovarian follicles Giuseppe C. Balboni, M.D. t Gabriella B. Vannelli, M.D. t Tullio Barni, M.D.* Claudio Orlando, B.Sc.* Mario Serio, M.D.* University of Florence, Florence, Italy Transferrin and somatomedin C receptors (TFr and SMCr) were studied in human ovaries by immunostaining using monoclonal antisera. The oocyte of evoluting follicles was intensely positive for both types of receptors, but this positivity decreases in large follicles. An evident positivity for both TFr and SMCr was shown in granulosa cells of evoluting follicles. However, not all of these cells were equally immunoreactive. An intense positivity was present in the developing thecal cells of early cavitary follicles as well as in thecal cells of follicles of medium and large size (>6 mm) and in some cells of involuting follicles in initial atresia. These results seem to demonstrate that TFr and SMCr are present in different cellular components of the human developing and early involuting follicles. Fertil Steril 48:796, 1987 Transferrin (TF) is the plasma protein devoted to iron transport. The transferrin receptor (TFr), a glycoprotein located on the membrane of proliferating cells in several tissues, is essential for the iron transport into the cell, where this ion is used for cellular growth and metabolism. In a previous study, we demonstrated that immunoreactive TF is present in Sertoli's cells of human seminiferous tubules and that TFr is detectable in spermatocytes and early spermatids. 1 TF was shown to be present in the follicular fluid of stimulated ovarian follicles,2 but the localization of receptors in human ovary was not studied until now. Somatomedin C (SMC), or insulin-like growth factor I (IGF-I), is a peptide, growth-hormone dependent, that plays an important role in promoting cell metabolism and proliferation. Received February 4, 1986; revised and accepted June 16, * Supported by the University of Florence. t Institute of Human Anatomy. * Department of Clinical Physiopathology, Andrology Unit. Reprint requests: Mario Serio, M.D., Dipartimento di Fisiopatologia Clinica, Unite. di Andrologia, Viale Morgagni 85, Firenze, Italy. 796 Balboni et al. Transferrin and somatomedin C re~eptors The demonstration of a specific somatomedin C receptor (SMCr) is rather important to discriminate the SMC activity from that of insulin and insulin-like growth factor II, which is less mitogenic and has a higher insulin-like activity. According to Adashi et al.,a SMC is an important factor acting on granulosa cell replication and differentiation in the human ovary. Moreover, SMC seems to cooperate with follicle-stimulating hormone (FSH) in the induction of rat granulosa cell aromatase activity and with LH for progesterone (P) production. SMC acts in vivo by paracrine and/or autocrine mechanism.' Conversely, Poretsky et al.,5 using a competitive binding technique, reported an uncertain evidence ofigf-i receptor in normal human ovaries. The aim of this research was to localize TFr and SMCr by immunohistochemical methods in the human ovarian follicles. MATERIALS AND METHODS Tissue Preparation Tissue specimens from human ovaries (16 ovaries of eight cycling women; age range, 16 to 18 years) were obtained at autopsy within 1 hour after Fertility and Sterility

2 : I traumatic death or removed during pelvic surgery. A written consent was obtained from patients or relatives. In this research, evoluting and involuting follicles have been considered. The ovarian follicles, according to their stage of development, have been classified into four groups: (1) primary and secondary follicles; (2) small «6 mm); (3) medium and large (>6 mm) cavitary follicles; and (4) involuting follicles in initial obliteration and cystic atresia. Because of the variable number of the different types of evoluting follicles normally found in the human ovary, our observations were made on a conspicuous number of primary and secondary follicles (more than 100), a smaller number (44) of medium and large cavitary follicles, and more than 30 cases of involuting (atresic) follicles. The identification of evoluting and involuting follicles was made on the basis of the histologic appearance,6 taking into account the following criteria: (1) histologic appearance of the oocyte and zona pellucida; (2) presence or absence of regressive phenomenona in the granulosa layer; (3) thickness and periodic acid-schiff (PAS) positivity of the follicular basal membrane; and (4) arrangement of the theca interna cells around the follicular basal membrane. Reagents Murine monoclonal antibody B3/25 (a 92121) to human TFr was obtained from Hybritech, Inc. (San Diego, CA). Murine monoclonal antibody to human SMCr (air-3) was obtained from The Wellcome Research Laboratories (Research Triangle Park, NC). Anti-mouse IgG peroxidase conjugate (A-2028) and human TF were obtained from Sigma Chemical Company (St. Louis, MO). 3,4,3',4',tetra -aminodiphenilhydrochloride (Diaminobenzidine) was obtained from BDH Chemical Ltd. (Poole, England). Human SMC (preparation I14) was obtained from Dr. Humbel (Biochemistry and Metabolic Unit, University of Zurich, Switzerland). Procedures The ovaries were fixed in Bouin's solution. Immunoperoxidase staining was performed as described by Sternberger/ according to the protocol previously described by Vannelli et au The working dilutions of antisera were 1:400 for anti-smcr and 1:1000 for anti-tfr. The working dilution of anti-mouse IgG peroxidase conjugate was 1:1000. Positive results were controlled by (1) incubation with a nonimmune serum; (2) incubation with IgG peroxidase-conjugate without preincubation with TFr and SMCr antisera; (3) different dilutions of specific immune sera (1:800, 1:1600, and 1:4000 for SMCr antiserum and 1:2000, 1:5000, and 1:10000 for TFr antiserum); and (4) preincubating with either TF (15 to 65 nm) or SMC (70 to 250 nm) before incubating with the specific antisera. No positivity was found in all control sections studied (n = 50); (see also Figs. 1b, Ie, 2b, 3b, 4b, and 4f). RESULTS Primary and Secondary Follicles Primary oocyte is intensely immunoreactive for TFr, as well as for SMCr in all the cases observed. An evident positivity also was present in several granulosa cells (Figs. la, 1c, and 3a). The adjacent control sections do not show any reactivity (Figs. 1b, Ie, and 3b). Cavitary Follicles of Small Size «6 mm) Oocytes maintain sharp positivity for TFr and SMCr. In the granulosa cells, the immunoreactivity seems to increase, probably in relation to the increasing number of cells. A quantitative evaluation showed that almost two thirds of granulosa cells in the investigated follicles were clearly positive. Concerning the developing theca interna, an evident immunoreactivity for receptors is present in differentiating cells of the outer layers, whereas only some fully differentiated theca interna cells are intensely positive (Figs. 3c and 3d). Cavitary Follicles of Medium and Large Size (>6 mm) The positivity of oocyte for TFr and SMCr was still evident in all cases, but it seemed to be decreased in comparison to the previous stages. The immunoreactive granulosa cells were scattered within the follicular wall with an apparent concentration in the cumulus. Of course, the number of immunoreactive cells in each follicle was variable in relation to different factors, such as the plane of the section and its thickness. In any case, in all the follicles considered, a number of granulosa cells showed evident immunoreaction for both TFr and SMCr. The majority of theca interna cells showed an intense positivity for receptors (Figs. 4a and 4c). Balboni et al. Transferrin and somatomedin C receptors 797

(c) Evoluting primary follicle. Primary oocyte is intensely immunoreactive for TFr. An evident positivity is present in several granulosa cells (original magnification was X410).

3 Figure 1 Transferrin receptors. (a) Evoluting follicle of small size «6 mm) treated with monoclonal antibody anti-human TFr (B3/25) (1:1000 dilution). Many granulosa cells are intensely positive (original magnification was X410). (b) Adjacent section incubated with nonimmune serum (hematoxylin counterstained, original magnification was X410). (c) Evoluting primary follicle. Primary oocyte is intensely immunoreactive for TFr. An evident positivity is present in several granulosa cells (original magnification was X410). (d) Early involuting follicle treated with monoclonal antibody anti-human TFr (B3/25) (1:1000 dilution). Part of the thecal cell appears intensely immunoreactive (original magnification was X350). (e) Control section of part c preincubated with TF (15 nm) before adding the TFr antiserum (hematoxylin counterstained, original magnification was X410). The adjacent control sections did not show any reactivity (Figs. 4b and 4f). Involuting Follicles In the developed theca layer of early involuting follicles, a large number of cells appeared intensely immunoreactive for TFr and SMCr (Figs. 2a and Id). The adjacent control section did not show any reactivity (Fig. 2b). In the theca layer of advanced atretic follicles, some positive cells were still present (Figs. 4d and 4e). 798 Balboni et ai. Transferrin and somatomedin C receptors DISCUSSION The results we obtained in the present study demonstrate that receptors for TF and SMC are present in ooplasma of primary oocytes. The immunoreactivity of the oocyte for TFr and SMCr is quite intense. This finding is not surprising because it has been demonstrated that SMC is able to stimulate deoxyribonucleic acid (DNA) synthesis in several cell types with or without cell replication. 4 Because the oocytes are arrested at the prophase of the first meiotic division, they cannot rep- Fertility and Sterility

Figure 2 Transferrin receptors. (a) Atretic follicle treated with monoclonal antibody antihuman TFr (B3/25) (1:1000 dilution). Several theca cells are stained (original magnification was X410).

ever, not all of these cells are equally immunoreactive. According to Adashi et al.

On the other hand, SMC might also synergize with FSH in the induction of rat granulosa cell aromatase activity.

4 Figure 2 Transferrin receptors. (a) Atretic follicle treated with monoclonal antibody antihuman TFr (B3/25) (1:1000 dilution). Several theca cells are stained (original magnification was X410). (b) Control section treated with monoclonal antibody antihuman TFr (B3/25) at low dilution (1:10,000). No reactivity is detectable (hematoxylin counterstained, original magnification was X41O). ever, not all of these cells are equally immunoreactive. According to Adashi et al.,3 IGF-I might act by stimulating granulosa cell replication and promoting differentiation, probably in an autocrine way. On the other hand, SMC might also synergize with FSH in the induction of rat granulosa cell aromatase activity.l2 The fact that, in our experiments, not all granulosa cells of human evoluting follicles present an evident immunoreactivity for TFr and SMCr may be due to a different functional cellular stage or to differences in the origin and function of granulosa cells. 6 l3 Consequently, the results of Poretsky et al. 5 may be explained: using a competitive binding technique, they were unable to demonstrate clear evidence of IGF-I receptors in human granulosa cells. As far as the presence of receptors in the thecal cells is concerned, two results should be noted. First, an intense positivity is present in the developing thecal cells of early cavitary follicles. These cells are located between the layer of the fully differentiated thecal cells and the theca externa layer, licate. Our features therefore may be interpreted as a demonstration that TF and SMC promote oocyte growth (from 9-25 to ~m) during the first stages of follicular maturation. 8 It has been shown that increased follicular and oocyte diameter are positively and rectilineary correlated until oocytes reach a maximal diameter (mean value, 80 ~m), which they achieve when the mean follicular diameter reaches 124 ~m.8 After oocyte growth is completed, follicular growth continues, so that diameter of preovulatory follicles ranges from 10 to 20 mm. 9 In this way, the decreased positivity for SMCr and TFr in the oocyte of cavitary follicles can be explained. It is not clear whether TF and SMC have any effects on the resumption of oocyte meiosis in the mature follicle. The observation that the immunoreactivity for these receptors decreases in the oocyte of large follicle seems to indicate that these growth factors are not involved in this process. Conversely, the Ovary Meiosis Inhibitor lo ll assures the inhibition of meiosis until the LH surge. An evident positivity for TFr and SMCr is shown by the granulosa cells of evoluting follicles. How- Figure 3 Somatomedin receptors (air-3). (a) SecondarY follicle treated with monoclonal antibody to human SMC receptor (air-3) (1:400 dilution). PrimarY oocyte and several granulosa cells are positive (original magnification was X660). (b) Control section treated with monoclonal antibody to human SMC receptor (air-3) at low dilution (1:4000) (original magnification was X660). (c) Immunohistochemical demonstration of SMC receptor in a cavitary follicle of medium size (>6 mm). Many granulosa and thecal cells are stained (original magnification was X165). (d) CavitarY follicle (>6 mm). Prim8rY oocyte and several granulosa cells are intensively positive (original magnification was X225). Balboni et al. Transferrin and somatomedin C receptors 799

Figure 4 Somatomedin receptors. (a) The immunostaining for SMC receptor shows a sharp positivity in granulosa and thecal cells (original magnification was X165).

The majority of granulosa and theca interna cells show an intense positivity (original magnification was X660).

5 Figure 4 Somatomedin receptors. (a) The immunostaining for SMC receptor shows a sharp positivity in granulosa and thecal cells (original magnification was X165). (b) Adjacent unstained control incubated with nonimmune serum (hematoxylin counterstained, original magnification was X165). (c) higher magnification of the same specimen of part a. The majority of granulosa and theca interna cells show an intense positivity (original magnification was X660). (d, e) Advanced atretic follicles after incubation with monoclonal antibody to human SMC receptor (air-3, 1:400 dilution). Some thecal cells are stained (original magnifications were X660, X560, respectively). (f) Control section of part a preincubated with SMC (70 nm) before adding SMCr antiserum (1:1000 dilution; original magnification was X165). where the differentiation from the perifollicular stroma cells into theca interna cells occurs. In this case, the expression of receptors for growth factors is probably related to the differentiation process. Second, the thecal cells of medium and large evo- 800 Balboni et al. Transferrin and somatomedin C receptors luting follicles, as well as some scattered thecal cells of involuting (atretic) follicles, show an evident immunoreactivity for the receptors we studied. In these stages of follicular life in which phenomena of cellular proliferation and differentiation Fertility and Sterility

6 in the theca interna layer are not very active, the expression of TFr and SMCr is probably connected to the steroidogenetic activity of the cells The reduction of the number of receptor positive cells in the theca layer of advanced atretic follicles is probably due to a morphologic and functional regression that occurs in thecal cells with progressing atresia. Concerning the site of origin of TF and SMC, some data seem to suggest a local production oftf. According to our recent studies, immunoreactive TF is present in granulosa as well as in thecal cells (data not shown). In our opinion, the more probable site of production of TF is localized in granulosa cells. In fact, their cellular equipment appears to be essentially devoted to protein synthesis. 15 I6 Therefore, TF might act with a paracrine and/or autocrine mechanism. The same mechanisms have been proposed for SMC. 4 Acknowledgment. We thank Steven Jacobs, M.D., The Wellcome Research Laboratories, for the generous gift of somatomedin C receptor antiserum. REFERENCES 1. Vannelli BG, Orlando C, Barni T, Natali A, Serio M, Balboni GC: Immunostaining of transferrin and transferrin receptor in human seminiferous tubules. Fertil Steril 45:536, Entman SS, Maxon WS, Bradley CA, Osteen K, Wentz AC: Transferrin concentration (TRF) in follicular fluid (FF) of stimulated ovarian cycles (Abstr 59). Fertil Steril 41:25S, Adashi EY, Resnick CE, D'Ercole AJ, Svoboda ME, Van Wyk JJ: Insulin-like growth factors as intraovarian regula- tors of granulosa cell growth and function. Endocr Rev 6:400, Underwood LE, D'Ercole AJ, Clemmonds DR, Van Wyk JJ: Paracrine function of Somatomedins. Clin Endocrinol Metab 15:59, Poretsky L, Grigorescu F, Seibel M, Moses AC, Flier JS: Distribution and characterization ofinsulin and insulin-like growth factor I receptors in normal human ovary. J Clin Endocrinol Metab 61:728, Balboni GC, Vannelli GB: La relaxine dans les follicules de l'ovaire. Bull Assoc Anat (Nancy) 67:149, Sternberger LA: The unlabeled antibody peroxidase-antiperoxidase (PAP) method. In Immunocitochemistry. New York, Wiley Medical Publication, John Wiley and Sons, 1979, p Lintern-Moore S, Peters H, Moore GPM, Faber M: Follicular development in the inphant human ovary. J Reprod Fertil 39:53, Peters H, Biskov HG, Himelstein-Braw R, Faber M: Follicular growth: the basic event ofthe mouse and human ovary. J Reprod Ferti145:347, Moor RM, Warnes GM: Regulation of meiosis in mammalian oocytes. Br Med Bull 35:199, Tsafriri A, Channing CP: An inhibitory influence of granulosa cells and follicular fluid upon porcine oocyte meiosis in vitro. Endocrinology 96:922, Adashi EY, Resnick CE, Svoboda ME, Van Wyk JJ: Somatomedin C synergizes with follicle-stimulating hormone in the acquisition of progestin biosynthetic capacity by cultured rat granulosa cells. Endocrinology 116:2135, Zecchi S, Repice F, Balboni GC: On the granulosa cells of ovarian follicles. II. Identification of different morphological patterns of granulosa cells in evolutive follicles. Boll Soc Ital Biol Sper 57:583, Hsueh AJW: Paracrine mechanism involved in granulosa cell differentiation. Clin Endocrinol Metab 15:117, Balboni GC: Histology of the ovary. In The Endocrine Function of Human Ovary, Edited by VHT James, M Serio, G Giusti. London, Academic Press, 1976, p Balboni GC: Morphological remarks on the steroidogenesis in the human ovary. Folia Morphol Czech (Prague) 25:46, 1977 Balboni et al. Transferrin and somatomedin C receptors 801

Anna Maria Fulghesu, M.D. Alessandro Caruso, M.D. Salvatore Mancuso, M.D.*

Anna Maria Fulghesu, M.D. Alessandro Caruso, M.D. Salvatore Mancuso, M.D.* FERTILITY AND STERILITY Copyright e 1992 The American Fertility Society Vol. 57, No.1, January 1992 Printed on acid-free paper in U.S.A. Human growth hormone enhances progesterone production by human luteal