Article Benefits of 8-bromo-guanosine 3,5 -cyclic monophosphate (8-br-cGMP) in human ovarian cortical tissue culture

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1 RBMOnline - Vol 8. No Reproductive BioMedicine Online; on web 20 January 2004 Article Benefits of 8-bromo-guanosine 3,5 -cyclic monophosphate (8-br-cGMP) in human ovarian cortical tissue culture Jennifer Scott is a doctoral student at the Karolinska Institutet in Stockholm, Sweden, with collaborations at Prince Henry s Institute of Medical Research in her hometown of Melbourne, Australia. Her research investigates the initial recruitment of human ovarian follicles with particular interest in members of the TGFβ superfamily. Together with her colleagues, she is working towards the development of an optimal in-vitro culture system for maturing follicles from cryopreserved ovarian tissue, thus creating a potential new method of human assisted reproduction. Ms Jennifer Scott Jennifer E Scott 1,2,3, Pu Zhang 1, Outi Hovatta 1 1 Karolinska Institutet, Department of Obstetrics and Gynaecology, Karolinska University Hospital Huddinge, S Stockholm, Sweden; 2 Prince Henry s Institute of Medical Research, PO Box 5152, Clayton, 3168 Victoria, Australia 3 Correspondence: Jennifer.Scott@klinvet.ki.se Abstract Guanosine 3,5 -cyclic monophosphate (cgmp) is an important intracellular second messenger in many cells of the body; however, its importance in the ovary is only now being realized. The effects of the cgmp analogue 8-bromo-guanosine 3,5 - cyclic monophosphate (8-br-cGMP) were tested on human ovarian follicle development and survival using an established tissue culture method. Ovarian biopsies were collected from 27 women (mean age 32 years) undergoing Caesarean sections or gynaecological operations. Tissue was cut into small pieces and cultured in the presence or absence of 5 mmol/l 8-brcGMP for 7 and 14 days. 8-br-cGMP enhanced the rate of follicle growth to the secondary stage after both 7 and 14 days of culture. Furthermore, it significantly improved the proportion of viable follicles when compared with control cultures. All cultured follicles reaching the secondary stage were smaller than those uncultured. This suggests that although granulosa cells were stimulated to proliferate and form a double layer, the cells themselves did not become larger as usually occurs with maturation and differentiation. Oestradiol production was greater in the 8-br-cGMP-containing cultures after 12 days compared with control cultures, presumably due to the concurrent increase in the proportion of secondary follicles. In the early stages of human ovarian tissue culture, 8-br-cGMP may be a necessary component as both a growth enhancer and survival factor. Keywords: cgmp, culture, follicles, in-vitro maturation, ovary Introduction Ovarian folliculogenesis is a complex process involving endocrine, paracrine and autocrine pathways. In particular, the early stages are poorly understood including the initiation of dormant primordial follicles into the growing pool and progression to primary and secondary follicles. To help identify the factors involved in this follicle recruitment process, an in-vitro system culturing primordial human ovarian follicles within their stromal tissue has been established (Hovatta et al., 1997; Hreinsson et al., 2002). However, during culture many ovarian follicles suffer atresia, indicating a need to further optimize this culture system. It has been well established that adenosine 3,5 -cyclic monophosphate (camp) is the major second messenger in the ovary, mediating gonadotrophin actions including steroid and peptide hormone production, proliferation of granulosa cells, induction of oocyte maturation, and luteinization of granulosa and theca cells (Conti, 2002; LaPolt et al., 2003). Recently guanosine 3,5 -cyclic monophosphate (cgmp) has been receiving attention, and together with nitric oxide (NO) is also believed important in the regulation of granulosa cell activity. cgmp and NO have been shown to influence follicle development, apoptosis, steroidogenesis and inhibin A production, in addition to ovulation and oocyte maturation (Chun et al., 1995, 1996; McGee et al., 1997; Sirotkin et al., 319

2 ; Chen et al., 2003). During certain phases of the ovulatory cycle, it has been demonstrated in hamsters (Hubbard, 1980) and rats (Ratner and Sanborn, 1980) that concentrations of cgmp are inversely regulated to camp within whole ovaries. This indicates that cgmp may replace the signalling role of camp for local ovarian factors when camp concentrations are low. High concentrations of cgmp activate protein kinase G, resulting in the phosphorylation of Ser and Thr residues in target proteins, the same residues targeted by the camp-activated protein kinase. Thus, cgmp may effectively substitute the intracellular messages and actions of camp. Recent studies have implicated cgmp as a survival factor, preventing rat granulosa cell and follicle atresia. These survival studies were short-term (24 48 h) and involved isolated preantral (McGee et al., 1997), early antral (Chun et al., 1996), or preovulatory (Chun et al., 1995) follicles. The anti-apoptotic cgmp effects were observed by a reduction in DNA fragmentation; however, the actual protective mechanisms and intracellular effects of cgmp have not been described. To date, no studies have been performed in cultures of human ovarian tissue, or the very early stage follicles including primordial to secondary stages. This study aimed to determine if cgmp in a human ovarian tissue culture system could improve follicle viability, preventing the loss of follicles to atresia. Materials and methods Patients Twenty-seven women aged years (mean 32.2 ± SD 5.2) donated small ovarian cortical biopsies following informed consent. Biopsies were collected during routine Caesarean sections (n = 18) or gynaecological laparoscopies (n = 9). Ethical approval was obtained from the human research ethics committee of the Karolinska Institutet. Tissue culture Tissue was collected into pre-equilibrated HEPES-buffered oocyte collection medium (Gamete-100; Vitrolife, Kungsbacka, Sweden) containing 10% human serum albumin (HSA; Pharmacia and Upjohn, Stockholm, Sweden), and transported immediately to the laboratory. The biopsy was immersed in collection media, cut into small pieces of 1 2 mm 3 using a scalpel and placed into prepared wells of a 24- well culture dish (Nunclon; Nunc, Roskilde, Denmark). Wells contained Millicell culture plate inserts (Millipore, Sundbyberg, Sweden) coated with 100 µl pre-diluted Matrigel extracellular matrix (Becton Dickinson, Stockholm, Sweden). Matrigel was diluted 1:3 in alpha minimal essential medium (αmem; Gibco Invitrogen, Lidingö, Sweden) and used to support tissue growth (Hovatta et al., 1997). Serum-free culture medium of 100 µl was placed inside the insert with a further 400 µl outside the insert in the well. Culture medium comprised αmem with 10% HSA and 0.5 IU/ml recombinant human FSH (Gonal-F; Serono Nordic, Solna, Sweden) (Wright et al., 1999). Medium was supplemented with 1% ITS-G (Gibco) to supply concentrations of 10 µg/ml insulin, 5.5 µg/ml transferrin and 6.7 ng/ml sodium selenite; and 0.5% antibiotic/antimycotic solution (Gibco) providing 50 IU/ml penicillin G, 50 µg/ml streptomycin sulphate and µg/ml amphotericin B. The presence or absence of the widely used cgmp analogue, 8-bromo-guanosine 3,5 -cyclic monophosphate (8-br-cGMP; Sigma-Aldrich, Stockholm, Sweden), was tested. Previous studies had demonstrated the diffusion, effectiveness and suitability of this specific analogue at a 5 mmol/l concentration in rat ovarian follicle cultures (Chun et al., 1995, 1996; McGee et al., 1997). Each biopsy specimen from all the 27 patients was equally divided into five groups; uncultured, 7-day culture with or without 8-br-cGMP, and 14-day culture with or without 8-br-cGMP. Tissue was cultured in a humidified incubator at 37 C in 5% CO 2. Medium was sampled from the well outside the insert (100 µl) and partially replenished with fresh media (100 µl, 3 drops inside the insert with remainder outside) every second day. Fixation and analysis Following culture, tissue was fixed for 12 h in Bouin s solution and then dehydrated in 70% ethanol before being embedded in paraffin. Uncultured tissue from the same patient served as the fresh control. Tissue was cut in serial sections (4 µm) and stained with haematoxylin and eosin, allowing morphological analysis of follicle development and viability. To confirm the status of the follicle, adjacent sections were analysed, however, only the counts from every 10th section were recorded for data analysis to prevent double counting of the same follicle. The development stages of the follicle were classified by the number of layers and shape of granulosa cells surrounding the oocyte, previously defined by Gougeon (1996). Briefly, primordial follicles were classified as those containing a single layer of flattened granulosa cells, primary follicles with one or more cuboidal granulosa cells and secondary follicles where at least part of the follicle had two or more layers of cuboidal granulosa cells. The presence of pyknotic granulosa cells, eosinophilia of the ooplasm and clumping of the chromatin material identified atretic follicles (Gougeon, 1986). To measure follicle and oocyte diameter, a digital image analysis system (Easy Image Mätning; Tekno Optik, Huddinge, Sweden) connected to an inverted microscope (Nikon; Tekno Optik, Huddinge, Sweden) was used. Measurements of both follicle and oocyte were made at the largest oocyte diameter, with two perpendicular diameters measured and averaged for each. Oestradiol radioimmunoassay Samples of spent culture media (100 µl) were collected every second day and stored at 20 C for later analysis. The concentration of oestradiol was measured in these samples using the Coat-A-Count Oestradiol radioimmunoassay (Diagnostic Products Corporation, Los Angeles, CA, USA), with both inter- and intra-assay variations less than 10%. Data analysis Experiments were performed 27 times with one set of cultures for each biopsy obtained. Proportions of follicle stages and viability within each patient sample were compared for calculation of overall means and standard errors. Statistical analyses were performed on these mean values and mean oestradiol concentrations using a one-way ANOVA followed by tests of least significant difference. Diameters of follicles

3 and oocytes were compared using the t-test. Significance was accepted at the 0.05 level. Results Follicle viability A total of 1527 follicles were analysed in this study. Both 8-brcGMP and control cultured tissue showed a significant reduction in the proportions of viable follicles when compared with the uncultured tissue control (near 100%, Figure 1, P < 0.05). After 7 days of culture, the presence of 8-br-cGMP in the medium demonstrated improved follicle viability in comparison to the control culture group. Within individual experiments, the increase in the proportion of viable follicles was observed in 22 of the 27 cultures. No further significant differences were observed from day 7 to 14 of culture. time points, the culture medium supplemented with 8-brcGMP promoted further progression of the follicles to the secondary developmental stage when compared with the control medium. Within individual experiments, the increase in the proportion of secondary follicles with 8-br-cGMP was observed in 21 of the 27 cultures (day 7), and 20 of the 27 cultures (day 14). No significant differences were found within the cultured tissue groups when comparing culture duration. Follicle development stages Morphological analysis demonstrated viable follicles in uncultured (Figure 2a) and cultured tissue (Figure 2b, c) at all developmental stages. No differences were apparent in the stromal morphology from cultured tissue when compared with uncultured tissue. Figure 3 illustrates the follicular developmental changes that occur with culture. The initial mean proportion of primordial follicles in the uncultured tissue was 75%, which was significantly reduced in all cultures to values between 14 and 30% (all P < 0.05). The proportions of primary and secondary follicles significantly increased in all cultured tissue compared with uncultured tissue (all P < 0.05). Differences between control and 8-br-cGMP treatments were observed at the secondary follicle development stage at both day 7 (45 versus 34%) and day 14 (51 versus 36%). At both Figure 1. Follicle viability within uncultured tissue and tissue cultured in the presence and absence of 5 mmol/l 8-br-cGMP for 7 and 14 days (*P < 0.05 compared with uncultured #P < 0.05 compared with control 7 day). Total number of follicles assessed in each of the groups: uncultured (n = 492), 7 day control (n = 274), 7 day 8-br-cGMP (n = 268), 14 day control (n = 288), 14 day 8-br-cGMP (n = 205). Figure 2. Collection of primordial follicles in uncultured tissue (a), primary follicle cultured for 14 days in the presence of 5 mmol/l 8-br-cGMP (b), and secondary follicle (left) with primary follicle (right) cultured for 14 days in control media (c). For all panels, original magnification 400, bar = 30 µm. 321

4 the significantly reducing number of dormant primordial follicles, even within the first 7 days of culture. With the activation of more of these primordial follicles and entrance into the growing pool, more are subjected to apoptosis of granulosa cells and atresia, a process that also occurs naturally in the ovary (McGee et al., 1997). It has been shown that adding 8-br-cGMP to the culture media reduces this effect, hence supporting its role in humans as a survival factor, identified previously in rodents (Chun et al., 1995, 1996; McGee et al., 1997). 322 Figure 3. Follicle development within uncultured tissue and tissue cultured in the presence and absence of 5 mmol/l 8-brcGMP for 7 and 14 days (all cultured tissue stages significant P < 0.05 compared with uncultured, *P < 0.05 compared with control 7 day secondary, #P < 0.05 compared with control 14 day secondary). Total number of viable follicles assessed in each of the groups: uncultured (n = 491), 7 day control (n = 142), 7 day 8-br-cGMP (n = 165), 14 day control (n = 149), 14 day 8-br-cGMP (n = 127). Follicle and oocyte diameters Follicle and oocyte diameter measurements revealed no differences between groups or duration of culture, except concerning the secondary follicles (Table 1). As demonstrated, secondary follicles in uncultured ovarian tissue are of a significantly larger size with larger oocytes when compared with secondary follicles in all cultured tissue. No difference exists, however, between the cultured groups. Oestradiol concentration The concentration of oestradiol increased in the culture media of both treated and control groups with time. Throughout the 14-day culture period, ovarian cortical tissue cultured with 8- br-cgmp consistently produced more oestradiol than tissue cultured in control media (Figure 4), with significance achieved at day 12 only. Discussion In-vitro culture of human ovarian cortical tissue may be improved by the presence of specific hormones and factors, including cgmp. It has been shown that the cgmp analogue 8- br-cgmp is beneficial in the early stages of culture, improving both follicle viability and enhancing development. The results demonstrate that 8-br-cGMP helps to reduce the degree of follicle atresia that normally occurs with culture due to increased activation and growth in many follicles. This is highlighted by The addition of 8-br-cGMP to the culture media also had a profound effect upon the development of the follicles. Follicles are spontaneously activated to grow when removed from the body and placed in culture, probably due to the removal of an unknown substance inhibiting follicle recruitment. For this reason, most follicles are released from their dormant resting state as primordial follicles to develop into primary and later secondary follicles (Hreinsson et al., 2002). This was clearly demonstrated by the significant reduction in the number of primordial follicles in both cultured groups at 7 days. Consequently, the proportions of primary and secondary follicles increased, further demonstrating the significant activation of follicle growth. After 7 days of culture, both control and 8-br-cGMP groups had similar proportions of primordial and primary follicles. However, tissue treated with 8- br-cgmp revealed further maturation, with significantly more follicles at the secondary stage. This indicates proliferation of granulosa cells resulting in their double layer surrounding the oocyte, defining the secondary follicle. A similar significant effect was observed at 14 days suggesting that cgmp is important in granulosa cell proliferation and hence follicle development within the first 2 weeks of human ovarian tissue culture, and possibly longer periods. Similar beneficial effects on follicle development and survival have also been observed in cultures with camp (unpublished observations). This further highlights the importance of these intracellular second messengers in the cells of the ovary, and may identify their potential role in treatments for bypassing receptor-mediated responses including FSH-receptor mutations. The enhanced granulosa cell proliferation is supported by the oocyte and follicle diameter measurements. All cultured follicles reaching the secondary stage were smaller in diameter than those uncultured. Although oocytes were also significantly smaller in cultured secondary follicles, this cannot completely account for the reduction in follicle diameter. It is postulated that the granulosa cells in the cultured tissue were stimulated to proliferate rapidly, rather than complete their maturation and differentiation to produce larger cells. This results in follicles with more granulosa cell layers and hence greater development stage according to the classifications used (Gougeon, 1996). These cultured follicles may therefore not function the same as in-vivo formed secondary follicles, requiring further growth factors such as activin A to promote their differentiation and functional capabilities (Yokota et al., 1997; Oktay et al., 2000). In accordance with the increasing proportions of later-stage follicles with culture time is the increase in oestradiol production. As there was no increase in follicle diameter between 7 and 14 days, it is suspected that there is no increase in the number of granulosa cells per follicle during the second week of culture. It is suggested that in the first 7 days of culture the granulosa cells activate and proliferate to form a double

5 Table 1. Diameter of secondary follicles and their oocytes within uncultured tissue and tissue cultured in the presence and absence of 5 mmol/l 8-br-cGMP for 7 and 14 days. Uncultured 7 day control 7 day cgmp 14 day control 14 day cgmp (n = 47) (n = 26) (n = 34) (n = 19) (n = 19) Oocyte diameter 43.4 ± ± 1.3 a 34.6 ± 1.3 a 37.1 ± 1.2 a 37.6 ± 1.5 a (µm), mean ± SEM Follicle diameter 83.2 ± ± 3.4 a 66.0 ± 3.4 a 72.4 ± 3.3 a 66.4 ± 2.6 a (µm), mean ± SEM a P < 0.05 compared with uncultured. Matsumi et al., 1998). In the rat, NO has been shown to affect steroidogenesis of granulosa cells both cgmp-dependently and independently, seen particularly by the reduction in FSHstimulated oestradiol production (LaPolt et al., 2003). The present results demonstrate the reverse in human cultures with oestradiol concentrations increasing, and hence may indicate a species difference in this particular pathway. In conclusion, these findings are useful for the further development of an optimal method of ovarian tissue culture in vitro and have improved understanding of the mechanisms involved in follicle maturation. This work has direct implications for human fertility and assisted reproductive techniques. Acknowledgements Figure 4. Concentration of oestradiol in sampled culture media from ovarian cortical tissue cultured in the presence or absence (control) of 5 mmol/l 8-br-cGMP for 7 and 14 days (*P < 0.05 compared with 12 day control). layer and secondary follicle. Then in the following 7 days of culture (day 7 14), the granulosa cells somehow mature or differentiate to produce more oestradiol without becoming larger cells individually. A similar effect has been observed in cultures of isolated preantral mice follicles (Nayudu et al., 2002), where high doses of FSH produced a progressive increase in oestradiol without a corresponding increase in follicle growth. In these cultures, the excessive FSH dose (above the antral-threshold dose) resulted in an increased proportion of tightly packed mural granulosa cells and hence no increase in follicle diameter, yet an altered balance of differentiation amongst the granulosa cells. Further investigations are needed on later stage human ovarian follicles to observe if the same effect is occurring with 8-br-cGMP. The steroidogenesis pathway involving cgmp begins with soluble and receptor-bound guanylyl cyclases that synthesize cgmp from its substrate GTP. The soluble form is activated by NO, which is produced itself by the NO synthase (NOS) enzyme. Inducible NOS (inos) has been identified as the predominant enzyme form in the granulosa cells of small immature rat ovarian follicles (Van Voorhis et al., 1995; From the Department of Obstetrics and Gynaecology, Karolinska University Hospital Huddinge we are grateful to the surgical staff for their enthusiastic participation and cooperation in collecting ovarian biopsies, Julius Hreinsson for his valuable help and advice, and Jan-Åke Agren and Kjell Carlström for performing the oestradiol radioimmunoassay. We also thank Eva Blomén and Birgitta Siljestrand from the Department of Oral Pathology for the tissue sectioning and staining. The Swedish Medical Research Council and a scholarship from the Swedish Institute (J. E. S.) helped fund this study. References Chen Y-HJ, Tafoya MA, Ngo A et al Effects of nitric oxide and cgmp on inhibin A and inhibin subunit mrna levels from cultured rat granulosa cells. Fertility and Sterility 79, Chun SY, Eisenhauer KM, Kubo M et al Interleukin-1 beta suppresses apoptosis in rat ovarian follicles by increasing nitric oxide production. Endocrinology 136, Chun SY, Eisenhauer KM, Minami S et al Hormonal regulation of apoptosis in early antral follicles: follicle-stimulating hormone as a major survival factor. Endocrinology 137, Conti M 2002 Specificity of the cyclic adenosine 3,5 -monophosphate signal in granulosa cell function. Biology of Reproduction 67, Gougeon A 1986 Dynamics of follicular growth in the human: a model from preliminary results. Human Reproduction 1, Gougeon A 1996 Regulation of ovarian follicular development in primates: facts and hypotheses. Endocrine Reviews 17, Hovatta O, Silye R, Abir R et al Extracellular matrix improves survival of both stored and fresh human primordial and primary ovarian follicles in long-term culture. Human Reproduction 12,

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