Department of Biological Sciences University of Warwick Coventry CV4 7AL UK
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Abstract |
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Key words: antrum formation/follicle growth in vitro/FSH/in-vitro culture/mouse follicles
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Introduction |
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We have chosen to explore the gonadotrophin responsiveness of mouse follicles in the substrate-adherent system in order to gain information about follicle longevity and ovulatory efficiency in vitro. Such information may be of value in future for the culture of follicles from larger species, such as humans, where growth of intact follicles to full size (>20 mm diameter) is unlikely to be feasible in vitro.
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Materials and methods |
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The culture medium comprised minimal essential medium alpha (MEM; Gibco) containing 5% heat-inactivated fetal calf serum (FCS; Gibco) (or alternative sera as indicated below), penicillin (50 µg/ml), streptomycin (50 µg/ml) and apo-transferrin (10 µg/ml). FSH (HP Metrodin; Serono, Welwyn Garden City, UK) was prepared in unsupplemented culture medium and stored in aliquots at 20°C until used to produce final concentrations of 01000 mIU/ml.
Cultured follicles were observed daily to assess cellular attachment, growth, formation of antrum-like cavities, premature extrusion of oocyte and degeneration. Dimensions were measured only for follicles in which the basement membrane was intact and measurements were made of the area inside the basement membrane. Two perpendicular directions were measured using a previously calibrated eyepiece graticule on a dissecting microscope at a magnification of at least x800 and the mean was calculated and used for analyses. Once the basement membrane had ruptured, follicle appearance was described qualitatively. Follicles were defined as surviving when the oocyte was retained, appearing round in shape and follicle cells were not degenerate. HCG (Pregnyl; Organon, Oss, The Netherlands) was added to surviving follicles at a final concentration of 1.5 IU/ml and ovulation was noted 16, 24, 40 and 48 h later. Ovulation was defined as the complete release of the oocyte from the follicle after HCG addition. The presence or absence of attached cumulus cells was noted.
To assess viability, live and dead cells in some cultured follicles were detected by staining with carboxy fluorescein diacetate (CFDA; Sigma, Poole, UK) and propidium iodide (PI; Sigma, Poole, UK) respectively using a modification of a published method (Garner et al., 1986). CFDA (Sigma) was prepared as a stock solution of 0.46 mg/ml in phosphate-buffered saline (PBS), stored in aliquots at 20°C wrapped in foil and was used at a 1:50 dilution to give a final concentration of 9.2 µg/ml. PI was prepared as a stock solution of 1 mg/ml in PBS, stored at 4°C and was used at a 1:25 dilution to give a final concentration of 40 µg/ml. Follicles in situ in culture were washed in serum-free culture medium (MEM
) and medium containing CFDA was added. This was incubated at 37°C for 2030 min and then the solution was carefully removed. The follicle was washed with serum-free culture medium (MEM
) and then PI-containing medium was added. After 5 min at room temperature, PI was removed and replaced with serum-free culture medium (MEM
). The follicles were viewed using an inverted fluorescence microscope (Nikon) equipped with rhodamine, fluorescein isothiocyanate and triple filters (to observe staining with PI, CFDA and both respectively).
The following experiments were undertaken:
Log doseresponse curve to FSH (n = 322 follicles)
Mouse follicles were cultured in the presence of 0, 10, 100 and 1000 mIU/ml FSH. The day upon which antrum formation was first observed was noted. HCG was added on day 12 and ovulation noted at 16, 24, 40 and 48 h post HCG addition.
Effect of protein source (n= 180 follicles)
Follicles were cultured in the presence of six different protein sources for 12 days in the presence of 100 mIU/ml FSH. HCG was added on day 12 and ovulation noted at 16, 24, 40 and 48 h post HCG addition. The protein sources used were as follows: human serum albumin (HSA, 1%; Immuno Ag, Vienna, Austria); mouse serum (5%, two different batches, MS2 and MS7); FCS (5%, Gibco); embryo stem cell tested FCS (ESFCS, 5%; Gibco); hypogonadal mouse serum (hpgMS, 5%, a kind gift from Prof. H.Charlton, Oxford). Sera not already treated were heat-inactivated by incubation at 56°C for 40 min.
Day of HCG addition (n= 651 follicles)
Follicles were cultured in the presence of 100 mIU/ml FSH and 5% FCS. HCG was added on day 9, 10, 11, 12 or 14. Ovulation was noted at 16, 24, 40 and 48 h post HCG.
Oocyte growth (n= 200 follicles)
Oocyte diameter was measured throughout culture to ovulation (HCG day 10) without disruption of the follicles. These measurements were approximate as it was not possible to distinguish the exact boundary of the oocyte through the surrounding cumulus cells. Measurements were the average of two perpendicular directions. Oocyte diameters were also measured for follicles with or without HCG addition on days 9, 10, 11 and 12. These measurements were made using an inverted microscope, after stripping off any cumulus cells, and included the zona pellucida.
For follicle survival and ovulation, 2x2 contingency tables with 2-test were performed. Where a mean and SD are quoted (such as for follicle and oocyte diameter), Student's t-tests were performed for normally distributed data. Unless otherwise stated, data are expressed as mean ± SD and P < 0.05 was considered significant.
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Results |
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Follicular antrum formation was significantly (P < 0.001) impaired when follicles were cultured with 0, 10 or 1000 mIU/ml FSH compared with 100 mIU/ml FSH (Figure 5). The antral cavities that were formed in the absence of FSH had collapsed by the end of culture, suggesting that the follicles could not sustain antral development without FSH. The starting diameter of follicles that formed antra without FSH present was 151.6 ± 8.8 µm, which is greater than the mean diameter of follicles cultured without FSH. It is also greater than the mean diameter (136.5 ± 17.2 µm) of follicles cultured with 100 mIU/ml FSH which formed antra in vitro. A concentration of 100 mIU/ml FSH was used for subsequent experiments.
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In hpgMS, ESFCS and FCS, follicular cells attached to the culture dish, with follicles developing antra and ovulating in response to HCG. The follicles appeared visibly similar during culture and the survival rates were not significantly different in the different sera (8793%, NSD); however, the ovulation rates varied, as shown in Figure 6. A significantly lower proportion of follicles cultured with hpgMS was able to respond to the ovulatory stimulus than in ESFCS (P < 0.05)
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A total of 651 follicles of initial diameter 139.0 ± 18.5 µm on day 0 was cultured. The survival rate was significantly (P < 0.001) lower on day 14 (74%) than on days 912 (8795%) as shown in Figure 7. The proportion of follicles ovulating also varied with the duration of culture. Follicles with HCG added on day 11 or later had a significantly (P < 0.010.001) reduced proportion of follicles with timely ovulation (within 24 h of HCG) compared with days 9 or 10. Few follicles that ovulated late (>24 h after HCG) had cumulus cells attached, irrespective of the day of HCG addition. However, 92% of follicles that ovulated within 24 h of the HCG stimulus had mucified cumulus cells attached. Cumulus cell attachment depended on the day of HCG addition. A significantly (P < 0.001) higher number of follicles had mucified cumulus cells attached to the oocytes when ovulating within 24 h of HCG addition on days 9 (99%), 10 (97%), 11 (95%) and 12 (86%), compared with day 14 (36%). A proportion of follicles (ranging from 19% to 32%) did not ovulate when HCG was added on day 914.
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Discussion |
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Our culture system is based upon an established method (Cortvrindt et al., 1996), but there are several notable differences in our approach. The mice used here were 28 days old in comparison with 14 days. We selected older mice, approaching puberty to reduce the potential confounding influence of sexual immaturity which can have a variety of effects on oocyte and follicle function (Bao et al., 2000
; Ledda et al., 2001
). We omitted insulin from our cultures since our preliminary experiments showed it to have no beneficial effect, and others have demonstrated interactions between insulin and FSH to the detriment of mouse follicle and oocyte development in vitro (Latham et al., 1999
). We used a highly purified urinary preparation of FSH instead of recombinant FSH because of uncertainty about the physiology of different isoforms in the artificial preparation (Yding Andersen et al., 1999
). Urinary human FSH has since been shown to have a slightly higher potency than recombinant human FSH on mouse follicle growth in vitro (Liu et al., 2000a
).
In this study, we monitored ovulation at 16, 24, 40 and 48 h after a fixed maximal dose of 1.5 IU/ml HCG. This dose was presumed maximal since 1 IU/ml was found to be a maximal ovulation-inducing stimulus in a non-adherent system for mouse follicle culture (Rose et al., 1999). In vivo, ovulation occurs 1214 h post HCG (Hogan et al., 1986
; Yoshimura and Wallach, 1987
). In some cultured follicles, ovulation was delayed until
24 h after HCG, possibly indicating suboptimal pre-HCG development. The significance of late ovulation is not known. Since ovulation in vivo occurs promptly, we thought that ovulation within 16 h indicated good HCG responsiveness, and anything later to be potentially indicative of suboptimal follicular development. The proportion of follicles showing this delayed response varies with the culture conditions or is evident from the illustrations (e.g. Figures 3, 6 and 7
) in which the timings of ovulation are indicated. Many late ovulated oocytes had reduced or no cumulus surrounding, indicative of a loss of follicle/oocyte communication and potentially associated with suboptimal growth or post-maturity. Our data showed that prolonged culture beyond 12 days was associated with a significantly lower survival rate; however, the timely ovulation rate had begun to decline even from day 9 (Figure 7
), suggesting that ovulation timing is a more effective indicator of follicle viability than survival in culture per se. Cortvrindt et al. (1996) assessed the response to a dose of HCG combined with epidermal growth factor (EGF) over a similar period of time in terms of cumulus cell mucification. They concluded that after day 12 (days 14 and 16) there was a significant reduction in the ability of cumulus cells to mucify. This may have related to the onset of spontaneous luteinization on day 14, although antral development continued and germinal vesicle arrest was maintained in oocytes until day 16. However, when oocyte maturation was used as the end-point, they determined days 12 and 14 to be optimal for the HCG/EGF stimulus, whereas we have shown that the chances of timely ovulation in response to HCG have already declined substantially by day 12 (Figure 7
). EGF is known to improve oocyte maturation and cumulus mucification in response to gonadotrophins in vitro (Boland and Gosden, 1994
; Smitz et al., 1998
), which may have contributed to this difference. These data show that aspects of granulosa cell function may be compromised by extended in-vitro development before the ovulatory stimulus.
The response to HCG requires the presence on granulosa cells of LH receptors, which are known to be stimulated by the action of FSH, indicating the importance of appropriate priming. The log doseresponse curve, showing the influence of FSH priming upon ovulation in vitro, revealed that high as well as low concentrations of FSH were suboptimal. Our optimal dose of 100 mIU/ml conforms with that found by Nayudu and Osborn (1992) who measured growth in non-attached cultured follicles over a 6 day period. While it is already known that absence of FSH is incompatible with normal follicle growth in vitro (Qvist et al., 1990; Nayudu and Osborn, 1992
; Cortvrindt et al., 1996
), an upper threshold for optimal FSH priming in the substrate-adherent system has not previously been ascertained. However, it might be expected that excessive exposure to gonadotrophins would result in receptor down-regulation, potentially leading to a suboptimal follicular response (LaPolt et al., 1992
).
Antrum formation in vivo begins at a diameter of ~200 µm (Spears et al., 1994). In this culture system, antrum formation was not evident until after the follicles had attached to the substrate and lost their three-dimensional shape, so a direct comparison with in-vivo conditions is not possible. Under suboptimal FSH concentrations, antrum formation in vitro was limited (Figure 5
), including when excess FSH was present. Cumulus cell function is known to be compromised by excess FSH causing inappropriate formation of LH receptors (Eppig et al., 1998
), however, it is uncertain whether cumulus cells may play a role in the formation of the antrum, as the mechanism remains unclear. Without FSH, only transient antra were observed, persisting for a maximum of 2 days and collapsing by day 9. This probably indicates carryover of FSH effect from the in-vivo situation. Such carryover appears likely since Nayudu and Osborn (1992) found that antrum formation did not occur in the absence of FSH, but when FSH was added to the culture medium during the first 2 days only, the majority of follicles continued to grow and some formed antra.
In our series, when 100 mIU FSH was present, antrum formation was first evident on day 5, which is earlier than its initiation on day 8 observed by Cortvrindt et al. (1996). Also, they did not observe antrum formation in follicles cultured without FSH, in contrast with our data showing that some follicles lacking FSH supplementation formed antra transiently. This difference may relate either to the difference in size range of follicles cultured (85145 µm compared with 95205 µm in this study). Alternatively, the potential difference in potency of FSH supplements (Liu et al., 2000a) or their isoform mixtures may have had some effect. It is known that the least acidic FSH isoforms cause accelerated antrum formation in vitro relative to the more acidic forms (Vitt et al., 1998
), but their physiological relevance may be limited by their short half-life in vivo (Chappel, 1995
) or to a precise time around the late follicular phase (Yding Andersen et al., 1999
). Acidic forms are more prevelant in post-menopausal urine, the source of FSH used in this study, than serum (Wide and Wide, 1984
); however, urinary preparations and recombinant preparations of FSH appear to contain a similar profile of isoforms by isoelectric focusing (Chappel, 1995
). The optimal combination of isoforms for follicle development remains uncertain.
It is customary to include some form of protein supplement in culture media, often serum or a serum-derived protein(s) such as albumin. This is believed to serve several functions: buffering, binding of potentially toxic ions, nutrition, and potentially providing growth factors and other factors supportive of the cultured cells. However, the variability associated with sera especially is undesirable, and less complex alternatives such as albumin or synthetic replacements are sought. Our experiments have shown that supplementation with HSA would not support mouse follicle development in this culture system. This batch was in routine use for human IVF and known to be non-toxic. Most authors using adherent culture of mouse follicles employ FCS (Liu et al., 2000b), although rat follicles have been cultured in a substrate-adherent manner with BSA (Gore-Langton and Daniel, 1990
). Intact mouse follicle cultures are usually supplemented with 5% mouse serum (Spears et al., 1996
; Rose et al., 1999
).
Interestingly, neither of two batches of commercially available normal mouse serum (a mixture of male and female) as used previously for in-vitro follicle culture in a non-attached system (Hartshorne et al., 1994) supported follicle development here. In contrast, hypogonadal mouse serum, which had also been used previously in the spherical culture system (Hartshorne et al., 1993
), did support follicle growth in this series in the presence of supplementary FSH. Hypogonadal mice lack the GnRH gene and consequently have extremely low levels of FSH and LH. Their gonads remain permanently infantile unless exogenous FSH is administered (Mason et al., 1986
). This difference in follicle development in vitro between normal and hpg mouse serum, both supplemented with FSH in vitro, is interesting. It could relate to the levels of various other factors in the sera (for example steroids, other gonadotrophins such as LH, or binding proteins), or to the direct access to granulosa cells afforded by substrate-adherent follicles. Further investigation is needed to elucidate the key components causing the difference. While there was no difference in timely ovulation between hpg mouse serum and the two sources of FCS, more follicles remained unresponsive to the ovulatory dose of HCG, which may indicate that some factor is lacking in hpg serum which affects their priming. For example, androgens have positive effects upon intact follicle growth in vitro (Murray et al., 1998
).
Oocytes in cultured follicles grew from an initial diameter of 54 µm to a maximum diameter of 76 µm by day 9. The oocytes had already entered the growth phase before culture began, and as expected, follicle development continued after oocytes were fully grown. These results are slightly different from those of Cortvrindt et al. (1996) who found a shallower growth curve which continued throughout the full 16 days of their culture (Cortvrindt et al., 1996), although their starting and fully grown oocyte diameters were similar to ours. These final oocyte diameters approximate to those occurring in vivo, in contrast to other systems where the final diameter of cultured oocytes has been reported as smaller than normal (Eppig and Schroeder, 1989
; Eppig and O'Brien, 1996
). Developmental competence is not acquired until oocyte size exceeds a threshold of ~80% (Bar Ami and Tsafriri, 1981
) so size is an important measure of oocyte function. The differences in rates of growth could possibly relate to the different ages of the mice (Bao et al., 2000
). The exposure of fully grown oocytes to HCG did not affect their diameters. Oocyte growth requires gap junctional communication with granulosa cells to be maintained during follicle development. The production of fully grown oocytes is therefore an indication of the ongoing intercellular connections in a coordinated manner within the follicle structure, despite its attachment to the underlying substrate.
In conclusion, we have demonstrated the ability of mouse follicles to grow, form antra, and ovulate in vitro in an adherent culture system. The efficiency of these key markers of follicle function may be affected by the starting material and by the gonadotrophic support and culture conditions to which it is exposed. In our experiments, optimal results in terms of survival, follicular appearance and antrum formation were obtained with MEM supplemented with 5% heat-inactivated FCS and 100 mIU/ml FSH. This protocol resulted in survival of 87% of follicles to day 12. Addition of 1.5 IU/ml HCG on day 9 resulted in the greatest proportion (71%) of follicles ovulating within 16 h, indicating a prompt response to HCG, although statistically similar results were obtained for HCG addition on day 9.
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Acknowledgements |
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Notes |
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References |
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Submitted on July 27, 2001; accepted on January 2, 2002.