Abstract Isolated preantral follicles from prepubertal and adult tammars were cultured in defined (serum-free) media and follicle growth, antrum formation and follicle quality were compared. FSH promoted follicle growth and antrum formation in follicles from prepubertal tammars. FSH promoted growth of follicles from adults, but other factors present in serum were required for antrum formation. Therefore, once the hypothalamic-pituitary-gonadal axis is mature, hormones and growth factors must modify the mechanism of antrum formation in the tammar. Only follicles that developed an antrum in the presence of serum had differentiated granulosa and theca layers. Cumulus cells did not surround the oocyte in follicles that developed an antrum in serum-free conditions. Cumulus cells surrounded the oocyte of follicles that developed an antrum in the presence of serum, indicating that growth factors are required for granulosa differentiation and maintenance of oocyte-granulosa connections. Although granulosa differentiation and antrum formation occur at a similar time in vivo, they are independent processes in marsupials. While FSH stimulates follicle growth in vitro, granulosa and theca differentiation require more complex conditions to support germ cell development in marsupials. Folliculogenesis in vitro in Prepubertal and Adult Preantral Follicles from a Marsupial Nadine M. Richings, Susan M. Osborn, Geoffrey Shaw, Peter D. Temple-Smith and Marilyn B. Renfree Department of Zoology, The University of Melbourne, Victoria, 3010, Australia NMR: +613 8344 9745; n.richings@zoology.unimelb.edu.au; http://www.zoology.unimelb.edu.au/staff/richings.htm MBR: +613 8344 4376; m.renfree@zoology.unimelb.edu.au: http://www.zoology.unimelb.edu.au/staff/renfree.htm Introduction Female germ cells develop and mature within ovarian follicles. Antrum formation is the defining morphological feature of an antral follicle and granulosa differentiation invariably occurs with antrum formation in vivo in mammals [1]. Although early antral follicles develop in both prepubertal and adult mammals in vivo, later stage antral follicles only develop in adults. Although oogenesis and folliculogenesis begin in the fetus or neonate, the final stages of follicle and oocyte maturation only occur in the mature adult. From the first oestrous cycle, follicles are repeatedly exposed to hormones and growth factors that modulate their structure, function and response [2]. Preantral follicles in prepubertal animals have not been exposed to this environment, and this may result in differences in the responses of oocytes and follicles from prepubertal and adult animals when placed in culture. In vitro studies of folliculogenesis use culture systems that are species-specific. One of the consistent factors in studies of a particular species is the reproductive age of the follicle donor. Preantral follicles from prepubertal animals have generally been used for studies in the mouse [3], rat [4], sheep [5] and pig [6]. Preantral follicles from adult animals have been used in studies in the hamster [7] and human [8]. In the cow, preantral follicles from prepubertal [9] and adult [10] animals have been cultured in serum-free media containing growth factors, and only follicles from adults develop to antral stages in these conditions. Serum-free culture systems have been developed for the long-term (> 4 days) culture of preantral follicles from the hamster [7], human [8], rat [11] and cow [10]. These systems support growth and antrum formation and have been used to study apoptosis [11], the influence of ascorbic acid [12], extra-cellular matrix remodelling [13] and requirements for hormones and growth factors [7, 10]. Although folliculogenesis in vitro has been studied extensively in eutherians, there is only one report of follicle culture in a marsupial. Preantral follicles from prepubertal grey short-tailed opossums were cultured in a complex medium containing serum [14]. Although FSH promoted follicle growth through the 7 day culture period, no follicles developed to definitive antral stages. No studies have compared the development of follicles from prepubertal and adult mammals. The aim of this study was to examine and compare the in vitro development of preantral follicles from prepubertal and adult tammar wallabies (Macropus eugenii) in a serum-free culture system by assessing the growth of follicles, antrum formation and general follicle morphology. Figure 1 – For assessment, the continuum of antrum formation was divided into four categories: Preantral follicles show no sign of antrum formation. The first observable sign of antrum formation is when antral fluid begins to accumulate and shadow-like patches (P) are visible in the granulosa cell layer. As more antral fluid accumulates, small pockets of antral fluid accumulate into lacunae (L), and then the lacunae coalesce into a discernible antrum (A). Figure 2 – (a) Non-degenerate preantral follicles had an intact theca showing no bubbling, thinning or rupture and clear granulosa cells with no signs of degeneration. (b) Moderately degenerate follicles had a thinning or bubbling theca (B) and/or dark, granular granulosa cells. (c) Degenerate/ruptured follicles had a ruptured theca (R) and/or very dark and granular granulosa cells. (d) Follicle with ruptured theca and extruded oocyte (EO). scale bar = 100 µm. Materials and Methods Follicle Isolation and Culture Preantral follicles were: l mechanically dissected from excised ovaries of 6 prepubertal and 7 adult tammars. l randomly allocated to groups; only follicles with an intact theca and obvious oocyte were included. l cultured individually in 30 l drops of culture medium (Table 1) under mineral oil in 4-well culture dishes at 37°C in a humidified gas environment of 5% CO2 in air for 7 days. Prepubertal follicles: l used to examine the effect of FSH on follicle growth and development using defined, serum-free media (containing human serum albumin, HSA). l allocated to 1 of 3 groups: FSH-LOW, 0.75 IU/ml FSH; FSH-HIGH, 1.5 IU/ml FSH or FSH-0 (control group). Adult follicles: l used to compare growth and development in a defined medium (HSA) and in complex, undefined medium (fetal bovine serum, FBS). l The defined medium was the optimal serum-free medium determined from the prepubertal experiments (i.e. FSH-HIGH). Follicle Evaluation and Measurement l Follicles were examined at the start of culture and then daily. l The length and width of each follicle were used to calculate the arithmetic mean follicle diameter. l Growth was expressed as proportional increase in mean diameter during culture. l At the end of culture, antrum formation and follicle quality were assessed using an inverted microscope while the follicles were still in culture. l Antrum formation was divided into four categories: preantral, patches, lacunae or antrum (Figure 1). l Follicle quality was assessed as non-degenerate, moderately degenerate or degenerate/ruptured (Figure 2). l After culture, follicles were fixed in superfix, embedded in epoxy resin, sectioned at 1 m with a glass knife and stained with toluidine blue. Statistical Analysis Analysis of Variance (ANOVA): used to analyse the mean diameter changes in the follicle culture data. Chi Square Test: used to analyse categorical data (antrum formation, follicle quality) l Categories were combined for analysis when numbers in categories were too low to comply with the requirements of the Chi Square test. Antrum formation: patches + lacunae + antrum = antral signs Follicle quality: slightly degenerate + degenerate/ruptured = degenerate signs Results - Prepubertal Follicles Results - Adult Follicles Figure 3 - Preantral follicles from prepubertal tammars during and after culture in defined (serum-free) conditions. (a) Follicle cultured in FSH-LOW on Day 2 of culture and (b) on Day 7 of culture after a 20% increase in diameter and with a better developed theca. (c) Follicle cultured in FSH-LOW, on Day 7 of culture with antral patches (P) visible in granulosa layer and (d) after sectioning and staining the line of antral patches delineates better staining granulosa cells from poor-staining granulosa. Antral fluid (AF) is present between granulosa cells. (e) Follicle cultured in FSH-LOW that developed to early antral stage in culture. No cumulus cells are attached to the zona pellucida where the antrum has formed. The nucleus (N) of the oocyte indicates the reduced cumulus cell connection did not cause spontaneous meiotic resumption. (f) Follicle cultured in FSH-0 showing very poor granulosa and theca structure and many pyknotic nuclei in granulosa cells. Scale bar = 100 µm. Figure 5 - Preantral follicles from adult tammars during and after culture with human serum albumin (HSA) or fetal bovine serum (FBS). A follicle cultured with HSA had increased by 20% from (a) Day 0 to (b) Day 7. (c) Follicle cultured with HSA had well- formed granulosa and theca by Day 7 and had antral lacunae (L) which were obvious in (d) the stained section. Follicle cultured with FBS had increased in diameter by 43% from (e) Day 0 to (f) Day 7 and had well-formed theca but no antrum. (i) Follicle cultured with FBS had a well-formed early antrum with cumulus surrounding the oocyte and (j) well differentiated theca. MG=mural granulosa, MP=membrana propria, TI=theca interna, TE=theca theca externa. (k) Follicle cultured with FBS had an early antrum by Day 7, (l) with cumulus surrounding the oocyte and a well-formed theca. Scale bars = 100µm. Acknowledgments This work was supported by the Australian Research Council and an Australian Postgraduate Award to N.M.R. We thank the Wallaby Research Group for help with animal handling, Joan Clarke, Department of Zoology, The University of Melbourne, for sectioning and staining all fixed follicles and Dr Marie Herberstein, Department of Biological Sciences, Macquarie University, for statistical help and advice. References: 1. Peters H, McNatty KP. The Ovary. London: Granada Publishing; 1980. 2. Richards JS. Phys. Rev. 1980; 60: 51-89. 3. Nayudu PL, Osborn SM. J. Reprod. Fert. 1992; 95: 349-362. 4. Cain L, et al. Endocr. 1995; 136: 3369-3377. 5. Cecconi S, et al. Biol. Reprod. 1999; 60: 594-601. 6. Wu J, et al. Biol. Reprod. 2001; 64: 375-381. 7. Roy SK, Greenwald GS. J. Reprod. Fert. 1989; 87: 103-114. 8. Roy SK, Treacy BJ. Fert. Steril. 1993; 59: 783-790. 9. Wandji S-A, et al. Theriogen. 1996; 45: 817-832. 10. Gutierrez CG, et al. Biol. Reprod. 2000; 62: 1322-1328. 11. McGee E, et al. Endocr. 1997; 138: 2417-2424. 12. Thomas FH, et al. Reproduction 2001; 122: 487-495. 13. McCaffery FH, et al. Biol. Reprod. 2000; 63: 267-273. 14. Butcher L, Ullmann SL. Reprod. Fert. Dev. 1996; 8: 535-539. Figure 7 – Defined, serum-free conditions supported antrum formation in follicles from prepubertal tammars, but not in those from adults. This difference suggests that the mechanism of antrum formation is modified after maturation of the hypothalamic-pituitary-gonadal axis. When an antrum formed in vitro in follicles from prepubertal tammars, cumulus cells did not completely surround the oocyte, and the zona pellucida was directly exposed to the antral fluid. Preantral follicles from adult tammars developed to antral stages in vitro in the presence of serum. Cumulus cells surrounded the oocyte of these antral follicles and the theca had differentiated to theca interna and theca externa. Summary v Preantral follicles from tammars can grow and develop in vitro to early antral stages. v As in eutherians, FSH is essential for growth of preantral follicles in the tammar and for the preantral to antral transition. v FSH stimulates antrum formation in follicles from prepubertal animals, but once the hypothalamic-pituitary-gonadal axis is mature, the control of antrum formation is more complex. v Differentiation of granulosa and theca requires factors other than FSH. v Antrum formation can occur without granulosa differentiation. v There are clear differences in the follicles from prepubertal and adult tammars. View publication stats View publication stats