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Changes in homologous and heterologous gap junction contacts during maturation-inducing hormone-dependent meiotic resumption in ovarian follicles of Atlantic croaker
GENERAL AND COMPARATIVE
ENDOCRINOLOGY General and Comparative Endocrinology 131 (2003) 291–295 www.elsevier.com/locate/ygcen
Changes in homologous and heterologous gap junction contacts during maturation-inducing hormone-dependent meiotic resumption in ovarian follicles of Atlantic croaker ~o,a,b,* Goro Yoshizaki,c and Peter Thomasd Digbo Bolamba,a Reynaldo Patin a
d
Texas Cooperative Fish and Wildlife Research Unit, Texas Tech University, Lubbock, TX 79409-2120, USA b US Geological Survey, Texas Tech University, Lubbock, TX 79409-2120, USA c Department of Aquatic Biosciences, Tokyo University of Fisheries, Minato-ku, Tokyo 108-8477, Japan Marine Science Institute, The University of Texas at Austin, 750 Channel View Drive, Port Aransas, TX 78373-1267, USA Accepted 13 November 2002
Abstract Homologous (granulosa cell–granulosa cell) gap junction (GJ) contacts increase in ovarian follicles of Atlantic croaker (Micropogonias undulatus) during the early (first) stage of maturation, but their profile during the second stage [i.e., during maturationinducing hormone (MIH)-mediated meiotic resumption] is unknown. The profile of homologous GJ contacts during the second stage of maturation in croaker follicles was examined in this study and compared to that of heterologous (granulosa cell–oocyte) GJ, for which changes have been previously documented. Follicles were incubated with human chorionic gonadotropin to induce maturational competence (first stage), and then with MIH to induce meiotic resumption. The follicles were collected for examination immediately before and after different durations of MIH exposure until the oocyte had reached the stage of germinal vesicle breakdown (GVBD; index of meiotic resumption). Ultrathin sections were observed by transmission electron microscopy, and homologous and heterologous GJ contacts were quantified along a 100-lm segment of granulosa cell–zona radiata complex per follicle (three follicles/time/fish, n ¼ 3 fish). Relatively high numbers of both types of GJ were observed before and after the first few hours of MIH exposure (up to the stage of oil droplet coalescence). GJ numbers declined during partial yolk globule coalescence (at or near GVBD) and were just under 50% of starting values after the completion of GVBD (P < 0:05). These results confirm earlier observations that GVBD temporally correlates with declining heterologous GJ contacts, and for the first time in teleosts show that there is a parallel decline in homologous GJ. The significance of the changes in homologous and heterologous GJ is uncertain and deserves further study. Ó 2003 Elsevier Science (USA). All rights reserved. Keywords: Teleost; Atlantic croaker; Gap junctions; Ovarian follicle; Maturation
1. Introduction The growth and maturation of ovarian follicles depend on a carefully orchestrated communication between the somatic cells of the follicle and the oocyte. This communication is in part achieved through gap junctions (GJs). Gap junctions are aggregates of intercellular membrane channels made of connexin (Cx) protein that allow the direct exchange of substances smaller than 1200 Da between adjacent cells (Gooden* Corresponding author. Fax: 1-806-742-2946. E-mail address: [email protected] (R. Pati~ no).
ough et al., 1996; White and Paul, 1999). Homologous (granulosa cell–granulosa cell) and heterologous (granulosa cell–oocyte) GJs have been observed in the ovarian follicles of all vertebrate species examined. The process of LH-dependent ovarian follicle maturation has been divided into two stages for a number of teleost fishes (Pang and Ge, 2002; Pati~ no et al., 2001). In the first stage, the ovarian follicle acquires the ability to produce maturation-inducing hormone (MIH) and the oocyte becomes sensitive to MIH stimulation, whereas in the second stage, MIH induces the resumption of meiosis. In Atlantic croaker (York et al., 1993) and in red seabream (Pati~ no and Kagawa, 1999), the first stage
0016-6480/03/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0016-6480(03)00015-7
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of maturation is accompanied by an increase in heterologous and homologous GJ contacts and, in croaker follicles, increased levels of Cx32.2 mRNA are also observed (Chang et al., 1999, 2000; Yoshizaki et al., 1994). Heterologous GJ coupling declines during the second stage of maturation in ovarian follicles of Atlantic croaker (York et al., 1993; Yoshizaki et al., 2001) and Fundulus heteroclitus (Cerd a et al., 1993). However, there is currently no information for any teleost species concerning the pattern of change in homologous GJ during the second stage of follicular maturation. Among mammals, changes (decreases) in homologous GJs (between cumulus cells or between membrana cells) have been documented during the period of meiotic resumption preceding ovulation in some (e.g., Eppig, 1982; Isobe et al., 1998; Larsen et al., 1986, 1987; Racowsky utovsk et al., 1989) but not all (S y et al., 1993) species. To understand the maturational role of homologous GJs in the teleost ovarian follicle, it is necessary to determine their pattern of change during the entire period of maturation. Therefore, the primary objective of the present study is to determine if changes occur in homologous GJ contacts during MIH-dependent meiotic resumption (second stage of maturation) in ovarian follicles of Atlantic croaker. Heterologous GJ were also examined as a point of reference (York et al., 1993; Yoshizaki et al., 2001) and to allow comparisons between the patterns of change in the two types of GJ.
2. Materials and methods 2.1. Chemicals General laboratory chemicals, DulbeccoÕs modified EagleÕs medium (DME) and human chorionic gonadotropin (hCG) were obtained from Sigma Chemical. 17a,20b,21-Trihydroxy-4-pregnen-3-one (MIH of Atlantic croaker) was purchased from Steraloids, Inc. 2.2. Animals and tissue collection Atlantic croaker were collected in the fall near Port Aransas, TX and maintained in indoor circular tanks under standard conditions as previously described (Pati~ no and Thomas, 1990a). The weight range of the fish used in the present study was 57.4–82.9 g. For tissue collection, fish were killed by spinal transection. Ovaries were removed and placed in DME supplemented with sodium bicarbonate (1.2 g/L), streptomycin sulfate (100 mg/L), and penicillin (60 mg/L) at pH 7.6. 2.3. Tissue cultures Ovarian lamellae were minced into small fragments. Approximately 30 mg of tissue was placed in each well
of 6-well culture plates containing 3 ml of DME with hCG (5 IU/ml) for 10–12 h to induce maturational competence. The follicles were then incubated with 290 nM MIH to induce meiotic resumption (Pati~ no and Thomas, 1990a). All incubations were carried out at 25 °C under gentle agitation. Separate experiments were conducted with tissue collected from three fish. Ovarian fragments were collected for analysis at 0, 60, 120, 240, and 480 min of MIH incubation for fish #1;at 0, 90, 150, 220, and 715 min for fish #2; and at 0, 90, 150, 240, and 330 min for fish #3.Times of sampling for each fish were adjusted to include various stages of meiotic resumption including germinal vesicle breakdown (GVBD). This was accomplished by visual inspection under a stereoscope of the cultures according to the criteria of Yoshizaki et al. (2001). 2.4. Electron microscopy Ovarian fragments were fixed with 2.5% glutaraldehyde in 0.1 M sodium cacodylate saturated with 1% lanthanum chloride (12–16 h at 4 °C). The samples were then rinsed in 0.1 M cacodylate buffer containing 1% lanthanum chloride and postfixed in 1% osmium tetroxide in 0.1 M cacodylate buffer. Following another buffer rinse, the fragments were dehydrated in a series of acetone and embedded in Epon (TAAB, Berks, England). Since the ovarian fragments contained a mixture of follicles at various stages of development, the stage of each follicle to be observed by electron microscopy was established by preliminary examination of semi-thin sections (approximately 0.5–1 lm) stained with 1% azur II and 1% methylene blue in 1% borax solution. Based on their morphology in semi-thin sections, ovarian follicles were classified into four categories: (I) maturationally competent follicle (small degree of oil droplet coalescence), (II) follicle with advanced oil droplet coalescence, (III) follicle with partial yolk globule coalescence, and (IV) follicle with complete yolk globule coalescence. The first two categories are preGVBD, the third category is at or near GVBD, and the fourth category is post-GVBD (Yoshizaki et al., 2001). We examined also the onset of germinal vesicle migration and presence or absence of the germinal vesicle, but these traits were not always visible in the section and thus were not used in the classification of follicles. Ultrathin sections (60–90 nm) were cut with a Diatome MT Ultra 35° diamond knife and an MT7061 ultramicrotome (Diatome, BIE/Bienne, Switzerland) and collected onto formvar-coated 100-mesh copper grids. Sections were stained with uranyl acetate and lead citrate, and viewed with a Hitachi HS9 electron microscope (Tokyo, Japan). The numbers of GJ contacts were determined along the equivalent of one grid length (100 lm) of the granulosa cell–zona radiata complex in each of at least three follicles per time (category) per fish.
D. Bolamba et al. / General and Comparative Endocrinology 131 (2003) 291–295
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Fig. 1. Heterologous (A) and homologous (B) gap junctions in ovarian follicles of Atlantic croaker. Note the presence of gap junction contacts (arrows); gcl, granulosa cell layer; ve, vitelline envelope. Magnification, 30,000. Scale bar, 0.2 lm.
2.5. Statistical analysis Not all follicles matured at the same pace. Thus, sampling time-associated data were re-classified according to the maturational category of the follicles examined (four categories were used; see preceding description). At least three follicles per category per fish were examined, and individual follicle values were averaged (within category) to obtain individual fish values per category. Statistical comparisons were conducted by using one-way ANOVA with follicle category as source of variation, followed by DuncanÕs multiple range tests. Differences were considered significant at P < 0:05.
conclusions. For those species where a decline in homologous GJ has been documented [mouse, Eppig (1982); rat, Larsen et al. (1986, 1987); pig, Isobe et al. (1998); hamster, Racowsky et al. (1989)], it has been hypothesized that the function of this decline is to interrupt the transfer of a maturation-inhibiting factor from the outer layers of granulosa (or membrana) cells
3. Results Homologous and heterologous GJs were observed in all follicles examined (Fig. 1). Maturationally competent follicles and follicles during advanced lipid coalescence had relatively high levels of both types of GJ (Fig. 2). Both types of GJ declined in follicles during partial yolk globule coalescence and became approximately one half of the initial values after complete yolk globule coalescence (P < 0:01; Fig. 2). The pattern of GJ change was similar when data for both types of GJ were combined for each follicle as an overall index of follicular coupling (Fig. 2).
4. Discussion The present study shows for the first time in teleost ovarian follicles that homologous GJs decline in parallel with the decline in heterologous GJs during the MIHdependent period of meiotic resumption. Therefore, the results obtained to date with Atlantic croaker (York et al., 1993; Yoshizaki et al., 2001; present study) indicate that the overall degree of GJ coupling (homologous and heterologous) increases in the ovarian follicle during the first stage of maturation, and then decreases late during the second stage. In mammals, studies of the role of homologous GJ during meiotic resumption have yielded contradictory
Fig. 2. Changes in individual and combined numbers of homologous and heterologous gap junction contacts during MIH-induced meiotic resumption in ovarian follicles of Atlantic croaker. Maturationally competent follicles were incubated in the presence of MIH to induce meiotic resumption, and they were sampled at various times until the occurrence of germinal vesicle breakdown. There was a time-dependent progression in maturation, but not all follicles matured at the same pace. Thus, time-associated data was reclassified according to maturational category of the follicles examined: (I) maturationally competent follicle (small degree of oil droplet coalescence), (II) follicle with advanced oil droplet coalescence, (III) follicle with partial yolk globule coalescence, and (IV) follicle with complete yolk globule coalescence. The first two categories are pre-GVBD, the third category is at or near GVBD, and the fourth category is post-GVBD (see text). The number of gap junction contacts was determined along a 100-lm segment of follicle wall by transmission electron microscopy. At least three follicles per category per fish were examined, and the examination was repeated with three different fish. Within each grouping (homologous, heterologous, or combined), bars associated with common letters are not significantly different (one-way ANOVA and DuncanÕs multiple range test, P < 0:05).
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to the oocyte, thus allowing oocyte meiosis to resume spontaneously. However, several research findings are inconsistent with this hypothesis. For example, contrary to the predictions of the hypothesis, a general disruption of GJ coupling had a suppressive effect, rather than an enhancing effect, on the in vitro resumption of meiosis in porcine follicles (Isobe et al., 1998). Further, in bovine follicles, homologous GJs do not decline until after utovsk GVBD has occurred (S y et al., 1993), and the premature uncoupling of homologous GJ suppresses GVBD (Vozzi et al., 2001). Unlike mammalian follicles, teleost ovarian follicles have a single layer of granulosa cells around the oocyte. Thus, the proposed roles of homologous GJ between inner and outer granulosa cell layers in the regulation of meiotic resumption in mammals (see preceding paragraph) is not applicable to teleosts. A different hypothesis has been recently proposed for teleosts on the basis of two observations. First, there is evidence in the general literature that GJs are involved in the regulation of steroidogenesis in granulosa cells (Fletcher and Greenan, 1985) and adrenocortical cells (Munari-Silem et al., 1995; Oyoyo et al., 1997; Shah and Murray, 2001). Second, the increase in homologous GJ observed during the first stage of maturation in ovarian follicles of Atlantic croaker (York et al., 1993) and red seabream (Pati~ no and Kagawa, 1999) correlates with the acquisition of their ability to produce MIH (Kagawa et al., 1991; Pati~ no and Thomas, 1990a). Thus, Pati~ no and Kagawa (1999) hypothesized that the ability to produce MIH is functionally associated with the increase in homologous GJ during the first stage of maturation. The results of the present study with Atlantic croaker are consistent with and extend this hypothesis, since the decline in homologous GJ that is observed late during the second stage of maturation (present study) is also associated with a concomitant decline in gonadotropin-dependent MIH production (Pati~ no and Thomas, 1990a,b). The present observation that heterologous GJs decline late during the second stage of maturation agrees with previous ultrastructural (York et al., 1993) and functional (Yoshizaki et al., 2001) studies with croaker follicles. The first noticeable decline in heterologous GJs in the present study occurred at the time of partial yolk globule coalescence, which in croaker follicles occurs near or at the time of GVBD and can be recognized by clearing of the cytoplasm (Yoshizaki et al., 2001). The late timing of this event and the observation that commitment to meiotic resumption occurs within minutes of MIH stimulation (Thomas and Das, 1997) suggest that, in croaker follicles, heterologous GJ uncoupling and the onset of meiotic resumption are correlative events that are not associated in a cause-effect manner. Further, it has been shown that post-GVBD croaker follicles can still maintain functional heterologous coupling (Yoshizaki et al., 2001).
In conclusion, the present results confirm earlier observations that GVBD temporally correlates with declining heterologous GJ contacts, and for the first time in teleosts show that there is a parallel decline in homologous GJ. Collectively, the information available for Atlantic croaker (York et al., 1993; Yoshizaki et al., 2001; present study) suggests that the overall level of GJ coupling (homologous and heterologous) in the ovarian follicle is at its height during the onset of MIH-dependent meiotic resumption and declines as the follicle completes the process of maturation and approaches ovulation. The significance of these changes in homologous and heterologous GJs is uncertain and deserves further study. Acknowledgments Funding for this work was provided by the US Department of Agriculture (NRICGP Animal Reproduction Grant #00-35203-9135) and the Japan Society for the Promotion of ScienceÕs Research for the Future program (97L00902). The authors would like to thank Ms. Susan Lawson and Drs. Mark Grimson, Vadim Salanikov, Izhar Khan, and Yong Zhu for their help.
References Cerda, J.L., Petrino, T.R., Wallace, R.A., 1993. Functional heterologous gap junctions in Fundulus ovarian follicles maintain meiotic arrest and permit hydration during oocyte maturation. Dev. Biol. 160, 228–235. Chang, X., Pati~ no, R., Thomas, P., Yoshizaki, G., 1999. Developmental and protein kinase-dependent regulation of ovarian connexin mRNA and oocyte maturational competence in Atlantic croaker. Gen. Comp. Endocrinol. 114, 330–339. Chang, X., Pati~ no, R., Yoshizaki, G., Thomas, P., 2000. Hormonal regulation and cellular distribution of connexin 32.2 and connexin 32.7 RNAs in the ovary of Atlantic croaker. Gen. Comp. Endocrinol. 120, 146–156. Eppig, J.J., 1982. The relationship between cumulus-oocyte coupling, oocyte meiotic maturation, and cumulus expansion. Dev. Biol. 89, 268–272. Fletcher, W.H., Greenan, J.R.T., 1985. Receptor mediated action without receptor occupancy. Endocrinology 116, 1660–1662. Goodenough, D.A., Goliger, J.A., Paul, D.L., 1996. Connexins, connexons, and intercellular communication. Annu. Rev. Biochem. 65, 475–502. Isobe, N., Maeda, T., Terada, T., 1998. Involvement of meiotic resumption in the disruption of gap junctions between cumulus cells attached to pig oocytes. J. Reprod. Fertil. 113, 167–172. Kagawa, H., Tanaka, H., Okuzawa, Y., Matsuyama, M., Hirose, K., 1991. Diurnal changes in plasma 17a,20b-dihydroxy-4-pregnen-3one levels during spawning season in the red seabream Pagrus major. Nippon Suisan Gakkaishi 57, 769. Larsen, W.J., Wert, S.E., Brunner, G.D., 1986. A dramatic loss of cumulus cell gap junctions is correlated with germinal vesicle breakdown in rat oocytes. Dev. Biol. 113, 517–521. Larsen, W.J., Wert, S.E., Brunner, G.D., 1987. Differential modulation of rat follicle cell gap junction populations at ovulation. Dev. Biol. 122, 61–71.
D. Bolamba et al. / General and Comparative Endocrinology 131 (2003) 291–295 Munari-Silem, Y., Lebrethon, M.C., Morand, I., Rousset, B., Saez, J.M., 1995. Gap junction-mediated cell-to-cell scommunication in bovine and human adrenocortical cells. A process whereby cells increase their responsiveness to physiological corticotropin concentration. J. Clin. Invest. 95, 1429–1439. Oyoyo, U.A., Shah, U.S., Murray, S.A., 1997. The role of a1 (connexin-43) gap junction expression in adrenal cortical cell function. Endocrinology 138, 5385–5397. Pang, Y., Ge, W., 2002. Gonadotropin and activin enhance maturational competence of oocytes in the zebrafish (Danio rerio). Biol. Reprod. 66, 259–265. Pati~ no, R., Kagawa, H., 1999. Regulation of gap junctions and oocyte maturational competence by gonadotropin and insulin-like growth factor-I in ovarian follicles of red seabream. Gen. Comp. Endocrinol. 115, 454–462. Pati~ no, R., Thomas, P., 1990a. Effects of gonadotropin on ovarian intrafollicular processes during the development of oocyte maturational competence in a teleost, the Atlantic croaker: evidence for two distinct stages of gonadotropin control of final oocyte maturation. Biol. Reprod. 43, 818–827. Pati~ no, R., Thomas, P., 1990b. Gonadotropin stimulates 17a,20b, 21trihydroxy-4-pregnen-3-one production from endogenous substrates in Atlantic croaker ovarian follicles undergoing final maturation in vitro. Gen. Comp. Endocrinol. 78, 474–478. Pati~ no, R., Yoshizaki, G., Thomas, P., Kagawa, H., 2001. Gonadotropic control of ovarian follicle maturation: the two-stage concept and its mechanisms. Comp. Biochem. Physiol. B 129, 427–439. Racowsky, C., Baldwin, K.V., Larabell, C.A., DeMarais, A.A., Kazilek, C.J., 1989. Down-regulation of membrana granulosa cell gap junctions is correlated with irreversible commitment to resume meiosis in golden Syrian hamster oocytes. Eur. J. Cell Biol. 49, 244–251.
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Shah, U.S., Murray, S.A., 2001. Bimodal inhibition of connexin 43 gap junctions decreases ACTH-induced steroidogenesis and increases bovine adrenal cell population growth. J. Endocrinol. 171, 199– 208. utovsky, P., Flechon, J.E., Flechon, B., Motlik, J., Peynot, N., S Chesne, P., Heyma, Y., 1993. Dynamic changes of gap junctions and cytoskeleton during in vitro culture of cattle oocyte cumulus complexes. Biol. Reprod. 49, 1277–1287. Thomas, P., Das, S., 1997. Correlation between binding affinities of C21 steroids for the maturation-inducing steroid membrane receptor in spotted seatrout ovaries and their agonist and antagonist activities in an oocyte maturation bioassay. Biol. Reprod. 57, 999–1007. Vozzi, C., Formenton, A., Chanson, A., Senn, A., Sahli, R., Shaw, P., Nicod, P., Germond, M., Haefliger, J.A., 2001. Involvement of connexin 43 in meiotic maturation of bovine oocytes. Reproduction 122, 619–628. White, T.W., Paul, D., 1999. Genetic diseases and gene knockouts reveal diverse connexin functions. Annu. Rev. Physiol. 61, 283– 310. York, W.S., Pati~ no, R., Thomas, P., 1993. Ultrastructural changes in follicle cell-oocyte associations during development and maturation of the ovarian follicle in Atlantic croaker. Gen. Comp. Endocrinol. 92, 418–420. Yoshizaki, G., Pati~ no, R., Thomas, P., 1994. Connexin messenger ribonucleic acids in the ovary of Atlantic croaker: molecular cloning and characterization, hormonal control, and correlation with appearance of oocyte maturational competence. Biol. Reprod. 51, 493–503. Yoshizaki, G., Pati~ no, R., Thomas, P., Bolamba, D., Chang, X., 2001. Effects of maturation-inducing hormone on heterologous gap junctional coupling in ovarian follicles of Atlantic croaker. Gen. Comp. Endocrinol. 124, 359–366.
ENDOCRINOLOGY General and Comparative Endocrinology 131 (2003) 291–295 www.elsevier.com/locate/ygcen
Changes in homologous and heterologous gap junction contacts during maturation-inducing hormone-dependent meiotic resumption in ovarian follicles of Atlantic croaker ~o,a,b,* Goro Yoshizaki,c and Peter Thomasd Digbo Bolamba,a Reynaldo Patin a
d
Texas Cooperative Fish and Wildlife Research Unit, Texas Tech University, Lubbock, TX 79409-2120, USA b US Geological Survey, Texas Tech University, Lubbock, TX 79409-2120, USA c Department of Aquatic Biosciences, Tokyo University of Fisheries, Minato-ku, Tokyo 108-8477, Japan Marine Science Institute, The University of Texas at Austin, 750 Channel View Drive, Port Aransas, TX 78373-1267, USA Accepted 13 November 2002
Abstract Homologous (granulosa cell–granulosa cell) gap junction (GJ) contacts increase in ovarian follicles of Atlantic croaker (Micropogonias undulatus) during the early (first) stage of maturation, but their profile during the second stage [i.e., during maturationinducing hormone (MIH)-mediated meiotic resumption] is unknown. The profile of homologous GJ contacts during the second stage of maturation in croaker follicles was examined in this study and compared to that of heterologous (granulosa cell–oocyte) GJ, for which changes have been previously documented. Follicles were incubated with human chorionic gonadotropin to induce maturational competence (first stage), and then with MIH to induce meiotic resumption. The follicles were collected for examination immediately before and after different durations of MIH exposure until the oocyte had reached the stage of germinal vesicle breakdown (GVBD; index of meiotic resumption). Ultrathin sections were observed by transmission electron microscopy, and homologous and heterologous GJ contacts were quantified along a 100-lm segment of granulosa cell–zona radiata complex per follicle (three follicles/time/fish, n ¼ 3 fish). Relatively high numbers of both types of GJ were observed before and after the first few hours of MIH exposure (up to the stage of oil droplet coalescence). GJ numbers declined during partial yolk globule coalescence (at or near GVBD) and were just under 50% of starting values after the completion of GVBD (P < 0:05). These results confirm earlier observations that GVBD temporally correlates with declining heterologous GJ contacts, and for the first time in teleosts show that there is a parallel decline in homologous GJ. The significance of the changes in homologous and heterologous GJ is uncertain and deserves further study. Ó 2003 Elsevier Science (USA). All rights reserved. Keywords: Teleost; Atlantic croaker; Gap junctions; Ovarian follicle; Maturation
1. Introduction The growth and maturation of ovarian follicles depend on a carefully orchestrated communication between the somatic cells of the follicle and the oocyte. This communication is in part achieved through gap junctions (GJs). Gap junctions are aggregates of intercellular membrane channels made of connexin (Cx) protein that allow the direct exchange of substances smaller than 1200 Da between adjacent cells (Gooden* Corresponding author. Fax: 1-806-742-2946. E-mail address: [email protected] (R. Pati~ no).
ough et al., 1996; White and Paul, 1999). Homologous (granulosa cell–granulosa cell) and heterologous (granulosa cell–oocyte) GJs have been observed in the ovarian follicles of all vertebrate species examined. The process of LH-dependent ovarian follicle maturation has been divided into two stages for a number of teleost fishes (Pang and Ge, 2002; Pati~ no et al., 2001). In the first stage, the ovarian follicle acquires the ability to produce maturation-inducing hormone (MIH) and the oocyte becomes sensitive to MIH stimulation, whereas in the second stage, MIH induces the resumption of meiosis. In Atlantic croaker (York et al., 1993) and in red seabream (Pati~ no and Kagawa, 1999), the first stage
0016-6480/03/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0016-6480(03)00015-7
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of maturation is accompanied by an increase in heterologous and homologous GJ contacts and, in croaker follicles, increased levels of Cx32.2 mRNA are also observed (Chang et al., 1999, 2000; Yoshizaki et al., 1994). Heterologous GJ coupling declines during the second stage of maturation in ovarian follicles of Atlantic croaker (York et al., 1993; Yoshizaki et al., 2001) and Fundulus heteroclitus (Cerd a et al., 1993). However, there is currently no information for any teleost species concerning the pattern of change in homologous GJ during the second stage of follicular maturation. Among mammals, changes (decreases) in homologous GJs (between cumulus cells or between membrana cells) have been documented during the period of meiotic resumption preceding ovulation in some (e.g., Eppig, 1982; Isobe et al., 1998; Larsen et al., 1986, 1987; Racowsky utovsk et al., 1989) but not all (S y et al., 1993) species. To understand the maturational role of homologous GJs in the teleost ovarian follicle, it is necessary to determine their pattern of change during the entire period of maturation. Therefore, the primary objective of the present study is to determine if changes occur in homologous GJ contacts during MIH-dependent meiotic resumption (second stage of maturation) in ovarian follicles of Atlantic croaker. Heterologous GJ were also examined as a point of reference (York et al., 1993; Yoshizaki et al., 2001) and to allow comparisons between the patterns of change in the two types of GJ.
2. Materials and methods 2.1. Chemicals General laboratory chemicals, DulbeccoÕs modified EagleÕs medium (DME) and human chorionic gonadotropin (hCG) were obtained from Sigma Chemical. 17a,20b,21-Trihydroxy-4-pregnen-3-one (MIH of Atlantic croaker) was purchased from Steraloids, Inc. 2.2. Animals and tissue collection Atlantic croaker were collected in the fall near Port Aransas, TX and maintained in indoor circular tanks under standard conditions as previously described (Pati~ no and Thomas, 1990a). The weight range of the fish used in the present study was 57.4–82.9 g. For tissue collection, fish were killed by spinal transection. Ovaries were removed and placed in DME supplemented with sodium bicarbonate (1.2 g/L), streptomycin sulfate (100 mg/L), and penicillin (60 mg/L) at pH 7.6. 2.3. Tissue cultures Ovarian lamellae were minced into small fragments. Approximately 30 mg of tissue was placed in each well
of 6-well culture plates containing 3 ml of DME with hCG (5 IU/ml) for 10–12 h to induce maturational competence. The follicles were then incubated with 290 nM MIH to induce meiotic resumption (Pati~ no and Thomas, 1990a). All incubations were carried out at 25 °C under gentle agitation. Separate experiments were conducted with tissue collected from three fish. Ovarian fragments were collected for analysis at 0, 60, 120, 240, and 480 min of MIH incubation for fish #1;at 0, 90, 150, 220, and 715 min for fish #2; and at 0, 90, 150, 240, and 330 min for fish #3.Times of sampling for each fish were adjusted to include various stages of meiotic resumption including germinal vesicle breakdown (GVBD). This was accomplished by visual inspection under a stereoscope of the cultures according to the criteria of Yoshizaki et al. (2001). 2.4. Electron microscopy Ovarian fragments were fixed with 2.5% glutaraldehyde in 0.1 M sodium cacodylate saturated with 1% lanthanum chloride (12–16 h at 4 °C). The samples were then rinsed in 0.1 M cacodylate buffer containing 1% lanthanum chloride and postfixed in 1% osmium tetroxide in 0.1 M cacodylate buffer. Following another buffer rinse, the fragments were dehydrated in a series of acetone and embedded in Epon (TAAB, Berks, England). Since the ovarian fragments contained a mixture of follicles at various stages of development, the stage of each follicle to be observed by electron microscopy was established by preliminary examination of semi-thin sections (approximately 0.5–1 lm) stained with 1% azur II and 1% methylene blue in 1% borax solution. Based on their morphology in semi-thin sections, ovarian follicles were classified into four categories: (I) maturationally competent follicle (small degree of oil droplet coalescence), (II) follicle with advanced oil droplet coalescence, (III) follicle with partial yolk globule coalescence, and (IV) follicle with complete yolk globule coalescence. The first two categories are preGVBD, the third category is at or near GVBD, and the fourth category is post-GVBD (Yoshizaki et al., 2001). We examined also the onset of germinal vesicle migration and presence or absence of the germinal vesicle, but these traits were not always visible in the section and thus were not used in the classification of follicles. Ultrathin sections (60–90 nm) were cut with a Diatome MT Ultra 35° diamond knife and an MT7061 ultramicrotome (Diatome, BIE/Bienne, Switzerland) and collected onto formvar-coated 100-mesh copper grids. Sections were stained with uranyl acetate and lead citrate, and viewed with a Hitachi HS9 electron microscope (Tokyo, Japan). The numbers of GJ contacts were determined along the equivalent of one grid length (100 lm) of the granulosa cell–zona radiata complex in each of at least three follicles per time (category) per fish.
D. Bolamba et al. / General and Comparative Endocrinology 131 (2003) 291–295
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Fig. 1. Heterologous (A) and homologous (B) gap junctions in ovarian follicles of Atlantic croaker. Note the presence of gap junction contacts (arrows); gcl, granulosa cell layer; ve, vitelline envelope. Magnification, 30,000. Scale bar, 0.2 lm.
2.5. Statistical analysis Not all follicles matured at the same pace. Thus, sampling time-associated data were re-classified according to the maturational category of the follicles examined (four categories were used; see preceding description). At least three follicles per category per fish were examined, and individual follicle values were averaged (within category) to obtain individual fish values per category. Statistical comparisons were conducted by using one-way ANOVA with follicle category as source of variation, followed by DuncanÕs multiple range tests. Differences were considered significant at P < 0:05.
conclusions. For those species where a decline in homologous GJ has been documented [mouse, Eppig (1982); rat, Larsen et al. (1986, 1987); pig, Isobe et al. (1998); hamster, Racowsky et al. (1989)], it has been hypothesized that the function of this decline is to interrupt the transfer of a maturation-inhibiting factor from the outer layers of granulosa (or membrana) cells
3. Results Homologous and heterologous GJs were observed in all follicles examined (Fig. 1). Maturationally competent follicles and follicles during advanced lipid coalescence had relatively high levels of both types of GJ (Fig. 2). Both types of GJ declined in follicles during partial yolk globule coalescence and became approximately one half of the initial values after complete yolk globule coalescence (P < 0:01; Fig. 2). The pattern of GJ change was similar when data for both types of GJ were combined for each follicle as an overall index of follicular coupling (Fig. 2).
4. Discussion The present study shows for the first time in teleost ovarian follicles that homologous GJs decline in parallel with the decline in heterologous GJs during the MIHdependent period of meiotic resumption. Therefore, the results obtained to date with Atlantic croaker (York et al., 1993; Yoshizaki et al., 2001; present study) indicate that the overall degree of GJ coupling (homologous and heterologous) increases in the ovarian follicle during the first stage of maturation, and then decreases late during the second stage. In mammals, studies of the role of homologous GJ during meiotic resumption have yielded contradictory
Fig. 2. Changes in individual and combined numbers of homologous and heterologous gap junction contacts during MIH-induced meiotic resumption in ovarian follicles of Atlantic croaker. Maturationally competent follicles were incubated in the presence of MIH to induce meiotic resumption, and they were sampled at various times until the occurrence of germinal vesicle breakdown. There was a time-dependent progression in maturation, but not all follicles matured at the same pace. Thus, time-associated data was reclassified according to maturational category of the follicles examined: (I) maturationally competent follicle (small degree of oil droplet coalescence), (II) follicle with advanced oil droplet coalescence, (III) follicle with partial yolk globule coalescence, and (IV) follicle with complete yolk globule coalescence. The first two categories are pre-GVBD, the third category is at or near GVBD, and the fourth category is post-GVBD (see text). The number of gap junction contacts was determined along a 100-lm segment of follicle wall by transmission electron microscopy. At least three follicles per category per fish were examined, and the examination was repeated with three different fish. Within each grouping (homologous, heterologous, or combined), bars associated with common letters are not significantly different (one-way ANOVA and DuncanÕs multiple range test, P < 0:05).
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to the oocyte, thus allowing oocyte meiosis to resume spontaneously. However, several research findings are inconsistent with this hypothesis. For example, contrary to the predictions of the hypothesis, a general disruption of GJ coupling had a suppressive effect, rather than an enhancing effect, on the in vitro resumption of meiosis in porcine follicles (Isobe et al., 1998). Further, in bovine follicles, homologous GJs do not decline until after utovsk GVBD has occurred (S y et al., 1993), and the premature uncoupling of homologous GJ suppresses GVBD (Vozzi et al., 2001). Unlike mammalian follicles, teleost ovarian follicles have a single layer of granulosa cells around the oocyte. Thus, the proposed roles of homologous GJ between inner and outer granulosa cell layers in the regulation of meiotic resumption in mammals (see preceding paragraph) is not applicable to teleosts. A different hypothesis has been recently proposed for teleosts on the basis of two observations. First, there is evidence in the general literature that GJs are involved in the regulation of steroidogenesis in granulosa cells (Fletcher and Greenan, 1985) and adrenocortical cells (Munari-Silem et al., 1995; Oyoyo et al., 1997; Shah and Murray, 2001). Second, the increase in homologous GJ observed during the first stage of maturation in ovarian follicles of Atlantic croaker (York et al., 1993) and red seabream (Pati~ no and Kagawa, 1999) correlates with the acquisition of their ability to produce MIH (Kagawa et al., 1991; Pati~ no and Thomas, 1990a). Thus, Pati~ no and Kagawa (1999) hypothesized that the ability to produce MIH is functionally associated with the increase in homologous GJ during the first stage of maturation. The results of the present study with Atlantic croaker are consistent with and extend this hypothesis, since the decline in homologous GJ that is observed late during the second stage of maturation (present study) is also associated with a concomitant decline in gonadotropin-dependent MIH production (Pati~ no and Thomas, 1990a,b). The present observation that heterologous GJs decline late during the second stage of maturation agrees with previous ultrastructural (York et al., 1993) and functional (Yoshizaki et al., 2001) studies with croaker follicles. The first noticeable decline in heterologous GJs in the present study occurred at the time of partial yolk globule coalescence, which in croaker follicles occurs near or at the time of GVBD and can be recognized by clearing of the cytoplasm (Yoshizaki et al., 2001). The late timing of this event and the observation that commitment to meiotic resumption occurs within minutes of MIH stimulation (Thomas and Das, 1997) suggest that, in croaker follicles, heterologous GJ uncoupling and the onset of meiotic resumption are correlative events that are not associated in a cause-effect manner. Further, it has been shown that post-GVBD croaker follicles can still maintain functional heterologous coupling (Yoshizaki et al., 2001).
In conclusion, the present results confirm earlier observations that GVBD temporally correlates with declining heterologous GJ contacts, and for the first time in teleosts show that there is a parallel decline in homologous GJ. Collectively, the information available for Atlantic croaker (York et al., 1993; Yoshizaki et al., 2001; present study) suggests that the overall level of GJ coupling (homologous and heterologous) in the ovarian follicle is at its height during the onset of MIH-dependent meiotic resumption and declines as the follicle completes the process of maturation and approaches ovulation. The significance of these changes in homologous and heterologous GJs is uncertain and deserves further study. Acknowledgments Funding for this work was provided by the US Department of Agriculture (NRICGP Animal Reproduction Grant #00-35203-9135) and the Japan Society for the Promotion of ScienceÕs Research for the Future program (97L00902). The authors would like to thank Ms. Susan Lawson and Drs. Mark Grimson, Vadim Salanikov, Izhar Khan, and Yong Zhu for their help.
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