Molecular Human Reproduction, Vol. 6, No. 6, 504-509,
June 2000
© 2000 European Society of Human Reproduction and Embryology
Ovary and oogenesis |
Spontaneous luteinization of antral marmoset follicles in vitro
Institute of Anatomy, Department of Cell Biology, E.M.A.-University, Friedrich Loeffler Strasse 23c, D-17487 Greifswald, Germany
Abstract
Large non-luteinized follicles of the marmoset monkey were cultured for up to 96 h in the presence of substances that are known to induce luteinization, i.e. LH, transforming growth factor (TGF)-ß and cyclic AMP. The state of the basal lamina, and the expression of connexin-43, 
2 integrin subunit and TGF-ß receptor type II (TßR-II) were chosen as parameters to judge the progress of luteinization. Antral follicles, cultured for 1 h, were not luteinized, as shown by an intact basal lamina, strong immunoreactivity of connexin-43 in granulosa cells, and no expression of TßR-II in the theca layer. After 12 h, most follicles showed a dissolution of the basal lamina, a faint reactivity of connexin-43, high expression of TßR-II in theca- and outer granulosa cells and high expression of 2 integrin subunit in granulosa cells bordering at the basement membrane; all of which indicate luteinization. After 96 h of culture, luteal structures (e.g. corpora lutea accessoria) had developed. This was true for both non-stimulated and stimulated follicles. Our results strongly suggest that antral follicles luteinize spontaneously. The decisive determinant appears to be the follicular stage.
follicle culture/luteinization/ovary/primate/transforming growth factors
Introduction
During folliculogenesis, cellular proliferation predominates in the early stages and differentiation predominates in the later stages. The differentiation during the later stages results either in luteal cells, or in cells undergoing atretic processes including apoptosis. The process of luteinization is thought to be under control of paracrine and endocrine factors like gonadotrophins and growth factors.
In-vitro studies using isolated granulosa or theca cells are of limited value, since luteinization depends on cross-talk between these two cell types in an intact follicle. Thus, the well-known phenomenon of spontaneous luteinization of cultured granulosa cells in vitro (Luck, 1994
) may be due to the lack of theca cells or of the basal lamina. We therefore used a culture system that permits cross-talk between the two follicular cell types to study the process of luteinization in vitro.
Studying luteinization means overcoming the problem of identifying luteal structures at the beginning of this process. In this study we used the dissociation of the basement membrane, the decrease of the gap junction protein connexin-43, the expression of a luteal specific member of the integrin family and the expression of transforming growth factor (TGF)-ß receptor type II (TßR-II) as markers of luteinization.
In antral follicles, the theca and granulosa cell layers are separated by a basement membrane. The basic components of this membrane are collagen IV and laminin (Yurchenco and Schittny, 1990; Paulsson, 1992). The follicular membrane may have a regulatory effect on granulosa cell proliferation and differentiation (Amsterdam et al., 1989; Richardson et al., 1992; Luck, 1994). Additionally, in healthy follicles it excludes capillaries, white blood cells and nerve processes from the granulosa compartment until ovulation, at which time the basal lamina is degraded (Rodgers et al., 1998). Thus, the degradation of the follicular membrane can be used as an early marker of luteinization.
Gap junctions are cell-to-cell contacts which facilitate the synchronization of follicular development. Connexin-43 is an ovarian gap junction protein (Schreiber et al., 1993; Mayerhofer and Garfield, 1995; Granot and Dekel, 1997; Lenhart et al., 1998) whose mRNA and protein concentrations increase during the oestrogen-dominated follicular phase, with highest expression in granulosa cells of large antral follicles (Schreiber et al., 1993; Mayerhofer and Garfield, 1995). During the initial phase of luteinization, connexin-43 mRNA and protein expression is down-regulated and remains low during this phase. Complete luteinized structures (e.g. corpora lutea accessoria) which develop later, display a very strong connexin-43 signal (Schreiber et al., 1993; Granot and Dekel, 1994). Thus, large antral follicles without connexin-43 expression must be those that luteinize.
Members of the TGF-ß family are potent stimulators of granulosa and theca cell differentiation (for review, see Shull and Doetschman, 1994). For example, TGF-ß1 stimulates progesterone production while inhibiting androgen production in theca cells (Magoffin et al., 1989) and promotes granulosa cell differentiation (Skinner et al., 1987). TGF-ß isoforms transduce a signal into the cell by binding to TßR-II. This initiates the formation of a heteromeric complex between TßR-II and TGF-ß receptor type I (TßR-I), with subsequent phosphorylation of the latter (Wrana et al., 1994). The receptor expression has turned out to be highly related to the process of luteinization (Wehrenberg et al., 1998).
As well as the criteria described for early luteinization, the expression of a member of the integrin family indicates more developed luteal structures. It has been shown (Giebel et al., 1996) that the expression of the 2 integrin subunit in large antral follicles is restricted to luteinized granulosa cells.
We used cultures of antral marmoset follicles to study the process of luteinization. The follicles were stimulated by application of either forskolin in combination with insulin or by LH alone or in combination with TGF-ß1. Luteinization of the follicles was identified by immunohistochemical analysis of collagen IV, connexin-43, TßR-II and 2 integrin. The in-vitro situation was then compared with the in-vivo situation.
Materials and methods
Animals
Marmoset monkeys (Callithrix jacchus) were maintained as previously described (Rune et al., 1992). The animals were killed by an overdose of thiopental (Byk van Gulden, Germany) and the ovaries were immediately removed. The cycle stage was determined morphologically according to previously described characteristics (Mansdotter et al., 1992; Wehrenberg et al., 1997).
Culture of antral follicles
Antral follicles, as examined by transillumination, were dissected from the ovaries with fine needles under a dissecting microscope. Follicles were collected in Dulbecco's modified Eagle's medium (DMEM; Biochrom, Germany) and washed several times in the same medium. The follicles were cultured in a double chamber system to maintain their morphological integrity according to a modified protocol (Trowell, 1959). The dissected follicles were placed on top of a collagen type I/type III-coated membrane (Transwell-Col, Costar, USA). The membrane was placed in a 6-well plate with each well containing 2 ml of serum-free DMEM. Under these conditions, the membrane just touched the surface of the medium and was not immersed in it. To stimulate luteinization in vitro the following substances were added to the medium from the beginning of cultivation. Insulin (100 ng/ml medium, Sigma, Germany) in combination with forskolin (4 µmol/l; Sigma), or human LH (250 ng/ml medium, Dr Parlow, UCLA Medical Center, USA) alone or in combination with recombinant TGF-ß1 (20 ng/ml medium; Genzyme, Germany). The concentrations used have been previously reported to stimulate luteinization: Insulin and forskolin (McArdle et al., 1991); LH (Engelhardt et al., 1992); TGF-ß (Roy, 1993). The follicles were cultured for up to 96 h. Following culture, the follicles were fixed in 4% paraformaldehyde at 4°C for 15 min and then frozen in liquid nitrogen.
Immunohistochemistry
A total of 150 antral follicles from 10 marmoset monkeys was used for immunohistochemistry. The frozen follicles were embedded in tissue tek (Miles, USA), cut in 7 µm sections on a cryostat (Cryostat 2800 Frigocut-N, Reichert-Jung, Germany) and mounted on silane-treated glass-slides. Additionally, cryo-sections of two marmoset ovaries were used for immunohistochemistry. The primary antibodies used were: rabbit polyclonal anti-TßR-II (Santa Cruz Biotechnology, USA), diluted 1:150 in phosphate-buffered saline (PBS); rabbit polyclonal anti-collagen IV (diluted 1:10 in PBS; Professor Merker, FU Berlin, Germany); mouse monoclonal anti-2 integrin (diluted 1:100 in PBS; Biomol, Germany); and mouse monoclonal anti-connexin-43 (diluted 1:200 in PBS; Dianova, Germany). Incubation with the TßR-II antibody was carried out overnight at 4°C. Incubation with the collagen IV, connexin-43 and 2-integrin antibodies was carried out for 2 h at room temperature. The specificity of the collagen IV antibody has been described previously (Rune et al., 1992). The binding of the collagen IV and connexin-43 antibodies was visualized with fluorecein isothiocyanate (FITC)-labelled secondary antibodies (1:200). Immunoreactivity of TßR-II and 2-integrin was localized using secondary antibodies (1:100) conjugated to alkaline phosphatase and stained with Fast Blue (0.12%; Dianova). Negative controls were done by omitting the primary antibody and by incubation with non-immune serum.
Results
Morphological characteristics of periovulatory ovaries
In the marmoset, as in other primates (Espey and Lipner, 1994), not only the dominant follicles, but also the theca cell layer of large follicles luteinize (Wehrenberg et al., 1997). This luteinization process results in the formation of corpora lutea accessoria. Thus, periovulatory ovaries of the marmoset typically consist of non-luteinized, small (preantral up to the beginning of antrum formation) follicles, luteinizing large follicles, atretic follicles and corpora lutea accessoria (Mansdotter et al., 1992; Wehrenberg et al., 1997, 1998).
Collagen IV expression
Collagen IV immunoreactivity was found in the basal lamina of all follicles in the periovulatory marmoset ovary. In vivo, the basal lamina of intact non-luteinized follicles (preantral up to the beginning of antrum formation) is apparent as a fine line between theca and granulosa cells (Figure 1a). Immunohistochemical visualization of the basal lamina with collagen IV revealed a sharp fine line in the non-luteinized follicles (Figure 1b). No immunoreactivity was found between granulosa cells. In large luteinized follicles the collagen IV immunosignal revealed a broad and wrinkled lamina and collagen IV was also localized in the granulosa cell layer (Figure 1c). Luteinization of theca and granulosa cells was determined morphologically (epitheloid shape of cells, enlargement of cells, increase of lipid droplets). In vitro, the basal lamina of cultured antral follicles, non-stimulated and cultured for 1 h (Figure 1d) appears as a fine line similar to that of non-luteinized follicles in vivo. No immunoreactivity was found between granulosa cells. After 12 h in culture (Figure 1e) a broad and wrinkled line between granulosa cells and theca cells appears, accompanied by a weak staining in the granulosa layer. After 96 h of culture the basal lamina of antral follicles, either non-stimulated (Figure 1f) or stimulated with insulin and forskolin (Figure 1g), has the identical shape of the lamina found in luteinized follicles in the ovary. Additionally, strong collagen IV reactivity was found between granulosa cells.
|
TßR-II expression
Immunoreactivity of the receptor was found in all luteinized cells of large follicles in vivo (Figure 2a
). Luteinization of theca cells and granulosa cells bordering the basal lamina was determined by morphological characteristics (see above). A few of the granulosa cells floating in the antrum are also immunoreactive. In-vitro; in freshly prepared antral follicles (Figure 2b) and in follicles at the beginning of culture, i.e. after 1 h (Figure 2c), the non-luteinized theca cells were immunonegative. After 96 h of culture, all luteinized cells of the follicles, whether stimulated or not, express TßR-II (Figure 2d–f). Comparison of the intensity of the immunosignal between non-stimulated follicles (Figure 2d), LH-stimulated follicles (Figure 2e) and insulin- and forskolin-stimulated follicles (Figure 2f) showed no differences.
|
Connexin-43 expression
In non-luteinized follicles, a strong connexin-43 immunoreactivity was observed (not shown). Only a weak connexin-43 expression was found in granulosa cells of follicles grown for 12 h on DMEM medium (Figure 3a
), and less expression was seen after 96 h (Figure 3b,c). Morphological examination of consecutive sections of these follicles revealed the typical characteristics of large luteinizing follicles. In insulin- and forskolin-stimulated follicles there was also no significant connexin-43 staining, but a few of the isolated and stimulated follicles had developed into a mature corpus luteum accessorium. These completely luteinized structures are connexin-43 positive (Figure 3d).
|
Expression of
2 integrin subunitIn all luteinized follicles, whether stimulated or not, the
2 integrin subunit is expressed in granulosa cells bordering the basement membrane. Granulosa cells floating in the antral cavity were immunonegative (Figure 4a).
|
Summary
The results concerning the luteinization of antral marmoset follicles are summarized in Table I
. Non-cultured follicles and follicles cultured for 1 h are not luteinized. After 12 h of culture, the number of luteinized follicles is increased, but not all follicles were luteinized. After 96 h of culture, all follicles were definitely luteinized. No differences between stimulated and non-stimulated follicles were observed. There was a small number of atretic follicles in all groups, but this did not vary with treatment.
|
Discussion
For our experiments we have chosen to use follicle cultures, since this provides a model system in which the interaction of the two follicular cell types that participate in luteinization remains intact. Thus, the response of both cell types to the addition of factors believed to stimulate luteinization in vitro could be studied. One study (Spears et al., 1998) showed that isolated follicles can be grown to the preovulatory stage. In the present study, we were able to show that isolated large antral follicles can luteinize in vitro up to the formation of luteal structures, e.g. corpora lutea accessoria. This was judged by the expression of collagen IV, connexin-43, TßR-II and 2 integrin subunit as markers of luteinization.
Within the follicle, theca and granulosa cells are separated by a basal lamina. The components of this basal lamina are collagen IV, laminin, fibronectin and heparan sulphate proteoglycan (for review, see Luck, 1994). Degradation of this lamina by collagenases is a requirement for the initiation of ovulation and subsequent luteinization. For example, inhibitors of mammalian tissue collagenases block ovulation in the perfused rat ovary (Brännström et al., 1988; Butler et al., 1991). Additionally, type IV collagenolytic activity present in human preovulatory follicular fluid increases prior to ovulation, and then rapidly declines as the follicles rupture (Puistola et al., 1986). In vivo, the rise in collagenases appears to be stimulated by LH within a few hours (Puistola et al., 1986; Luck, 1994). These results suggest that degradation of the basal lamina is an early event in the process of luteinization. Our in-vivo data confirmed these findings. The sharp belt-like collagen IV reactivity between theca and granulosa cells of intact non-luteinized follicles, indicating an intact basal lamina, is changed in follicles at the beginning of luteinization. These luteinized follicles displayed a disrupted collagen IV staining between theca and granulosa cells and additionally a staining within the granulosa cell layer. The same process of degradation was found in our culture system. After 1 h, the basal lamina of cultured antral follicles was still intact. After 12 h of culture the first signs of degradation were observed and by 96 h the basal lamina was completely degraded and collagen type IV reactivity was located within the granulosa layer. Thus, identical luteinization processes, as evidenced by the degradation of the basal lamina, take place in vivo and in vitro.
The fact that degradation of the basal lamina also appears in follicles undergoing atresia (Luck, 1994) does not interfere in these experiments, since visualization of pycnotic nuclei by transillumination allows us to discriminate between atretic and growing follicles. Moreover, luteinized follicles but not atretic follicles express 2 integrin subunit in granulosa cells bordering the basal lamina (Giebel and Rune, 1997), which was also the case in our study. Transillumination and 2 integrin subunit immunohistochemistry together guarantees that the cultured follicles are not atretic but are undergoing luteinization.
Similarly TßR-II is exclusively expressed in follicular cells starting to luteinize (theca cells and mural granulosa cells) and in completely luteinized cells of periovulatory marmoset ovaries (Wehrenberg et al., 1998). This dynamic of receptor expression was mirrored by our follicle culture system. At the beginning of culture, the theca cells were not luteinized and hence were TßR-II negative. At the end of culture (96 h), all theca cells and all mural granulosa cells were luteinized and accordingly, TßR-II expression was found in all cells.
Finally, the expression pattern of connexin-43 demonstrates that in our cultured follicles luteinization is initiated and maintained over the culture period. Before the formation of luteal structures in the early luteal phase in vivo, the gap junctions have been dissolved and, accordingly, the expression of connexin-43 is very low. When the formation of these luteal structures is accomplished and finally, completely luteinized structures have been developed, new gap junctions have been formed and, accordingly, the expression of connexin-43 is high (Schreiber et al., 1993; Mayerhofer and Garfield, 1995; Grazul-Bilska et al., 1996). The up- and down-regulation of connexin-43 expression that we found in vitro, is identical with that described for the in-vivo situation. Thus, at the beginning of luteinization, cultured large antral follicles showed only a weak connexin-43 expression which is completely down-regulated as luteinization progresses. Subsequently, completely luteinized structures (e.g. corpora lutea accessoria), display a very strong connexin-43 signal.
This study demonstrates that antral follicles luteinize in vitro, spontaneously and independent of any treatment. Our results strongly suggest that whole follicles are `programmed' to luteinize. Given this background, it appears that antral follicles are ripe to luteinize and can escape from this fate only by inhibitory factors.
In the context of stage-dependent luteinization, it is of special interest that antral follicles are ripe to luteinize spontaneously, whereas preantral follicles are not. This is confirmed by previous findings (Roy and Kole, 1998). They found that FSH and epidermal growth factor (EGF) stimulate the expression of TßR-II in human preantral follicles. The antral follicles used in our study are more developed and luteinize spontaneously, without any stimulation. Thus, factors which are known to stimulate luteinization, e.g. LH, FSH and EGF have no effect on these antral follicles.
Interestingly, the TßR-II expression level in isolated antral follicles of the marmoset was not affected by the ligand TGF-ß1 itself. This is also the case for preantral follicles of the human (Roy and Kole, 1998). Thus, the effect of TGF-ß on the differentiation of follicular cells seems not to depend on a positive feedback mechanism between the ligand and its receptor. TGF-ß receptor type I (TßR-I) was also unaffected by any stimulation and seems to be constitutively expressed in a majority of cells in the ovary (Roy and Kole, 1998).
In conclusion, our results demonstrate that antral follicles luteinize spontaneously. The decisive determinant is the follicular stage, rather than substances that are known to be supportive for luteinization. Luteinization seems to be a differentiation pathway that is programmed before antral formation.
Acknowledgments
The authors wish to thank Mrs H.Wegner for her excellent technical assistance. We are grateful to Professor H.J.Merker, Institute of Anatomy, Koenigin-Luise Strasse 15, D-14195 Berlin, Germany, for providing the antibody against collagen type IV. We thank Dr A.F.Parlow, National Hormone & Pituitary Program, Harbor-UCLA Medical Center, California 90509, USA, for kindly providing human LH.
Notes
1 To whom correspondence should be addressed at: Institute of Anatomy, Department of Cell Biology, E.M.A.-University, Friedrich-Loeffler-Str. 23c, D-17487 Greifswald, Germany. E-mail wehrenbe{at}rz.uni-greifswald.de 
References
Amsterdam, A., Rotmensch, S., Furman, A. et al. (1989) Synergistic effect of human chorionic gonadotropin and extracellular matrix on in vitro differentiation of human granulosa cells: progesterone production and gap junction formation. Endocrinology, 124, 1956–1964.[Abstract]
Brännström, M., Woessner, J.F., Koos, R.D. et al. (1988) Inhibitors of mammalian tissue collagenase and metalloproteinases suppress ovulation in the perfused rat ovary. Endocrinology, 122, 1715–1721.[Abstract]
Butler, T.A., Zhu, C., Mueller, R.A. et al. (1991) Inhibition of ovulation in the perfused rat ovary by the synthetic collagenase inhibitor SC 44463. Biol. Reprod., 44, 1183–1188.[Abstract]
Engelhardt, H., Tekpetey, F.R., Gore-Langton, R.E. et al. (1992) Regulation of steroid production in cultured porcine thecal cells by transforming growth factor-beta. Mol. Cell. Endocrinol., 85, 117–126.[ISI][Medline]
Espey, L.L. and Lipner, H. (1994) Ovulation. In E.Knobil and J.D.Neill (Eds) The Physiology of Reproduction. Raven Press, New York, USA, pp. 725–780.
Giebel, J. and Rune, G.M. (1997) Relationship between expression of integrins and granulosa cell apoptosis in ovarian follicles of the marmoset (Callithrix jacchus). Tiss. Cell, 29, 525–531.
Giebel, J., de Souza, P. and Rune, G.M. (1996) Expression of integrins in marmoset (Callithrix jacchus) ovary during folliculogenesis. Tiss. Cell, 28, 379–385.
Granot, I. and Dekel, N. (1994) Phosphorylation and expression of connexin-43 ovarian gap junction protein are regulated by luteinizing hormone. J. Biol. Chem., 269, 30502–30509.
Granot, I. and Dekel, N. (1997) Developmental expression and regulation of the gap junction protein and transcript in rat ovaries. Mol. Reprod. Dev., 47, 231–239.[ISI][Medline]
Grazul-Bilska, A.T., Redeemer, D.A., Johnson, M.L. et al. (1996) Gap junctional protein connexin 43 in bovine corpora lutea throughout the estrous cycle. Biol. Reprod., 54, 1279–1287.[Abstract]
Lenhart, J.A., Downey, B.R. and Bagnell, C.A. (1998) Connexin 43 gap junction protein expression during follicular development in the porcine ovary. Biol. Reprod., 58, 583–590.
Luck, M.R. (1994) The gonadal extracellular matrix. Ox. Rev. Reprod. Biol., 16, 33–85.
Magoffin, D.A., Gancedo, B. and Erickson, G.F. (1989) Transforming growth factor-ß promotes differentiation of ovarian thecal–interstitial cells but inhibits androgen production. Endocrinology, 125, 1951–1958.[Abstract]
Mansdotter, S., Epple, G. and Küderling, I. (1992) Age-related changes in ovarian morphology of the south american tamarin Sanguinus fuscicollis (Callitrichidae). J. Zool. Lond., 227, 1–19.
Mayerhofer, A. and Garfield, R.E. (1995) Immunocytochemical analysis of the expression of gap junction protein connexin 43 in the rat ovary. Mol. Reprod. Dev., 41, 331–338.[ISI][Medline]
McArdle, C.A., Kohl, C., Rieger, K. et al. (1991) Effects of gonadotropins, insulin and insulin-like growth factor I on ovarian oxytocin and progesterone production. Mol. Cell. Endocrinol., 78, 211–220.[ISI][Medline]
Paulsson, M. (1992) Basement membrane proteins: structure, assembly, and cellular interactions. Crit. Rev. Biochem. Mol. Biol., 27, 93–127.[ISI][Medline]
Puistola, U., Salo, T., Martikainen, H. et al. (1986) Type IV collagenolytic activity in human preovulatory follicular fluid. Fertil. Steril., 45, 578–580.[ISI][Medline]
Richardson, M.C., Davies, D.W., Watson, R.H. et al. (1992) Cultured human granulosa cells as a model for corpus luteum function: relative roles of gonadotrophin and low density lipoprotein studied under defined culture conditions. Hum. Reprod., 7, 12–18.
Rodgers, R.J., van Wezel, I.L., Rodgers, H.F. et al. (1998) Developmental changes in cells and matrix during follicle growth. In A.Lauri and F.Gandolfi (Eds) Gametes: Development and Function. Tabloid S.R.L., Rome Serono Symposia pp. 85–99.
Roy, S.K. (1993) Transforming growth factor-ß potentiation of follicle stimulating hormone induced deoxyribonucleic acid synthesis in hamster preantral follicles is mediated by a latent induction of epidermal growth factor. Biol. Reprod., 48, 558–563.[Abstract]
Roy, S.K. and Kole, A.R. (1998) Ovarian transforming growth factor-ß (TGF-ß) receptors: in vitro effects of follicle stimulating hormone, epidermal growth factor and TGF-ß on receptor expression in human preantral follicles. Mol. Hum. Reprod., 4, 207–214.
Rune, G.M., Leuchtenberg, U., Schröter-Kermani, C. et al. (1992) Zonal differentiation of the marmoset (Callithrix jacchus) endometrium. J. Anat., 181, 301–312.
Schreiber, J.R., Beckmann, M.W., Polacek, D. et al. (1993) Changes in gap junction connexin-43 messenger ribonucleic acid levels associated with rat ovarian follicular development as demonstrated by in situ hybridization. Am. J. Obstet. Gynecol., 168, 1094–1104.[ISI][Medline]
Shull, M.M. and Doetschman, T. (1994) Transforming growth factor-ß1 in reproduction and development. Mol. Reprod. Dev., 39, 239–246.[ISI][Medline]
Skinner, K.M., Keski-Oja, J., Osteen, K.G. et al (1987) Ovarian thecal cells produce transforming growth factor-ß which can regulate granulosa cell growth. Endocrinology, 121, 786–792.[Abstract]
Spears, N., Murray, A.A., Allison, V. et al. (1998) Role of gonadotropins and ovarian steroids in the development of mouse follicles in vitro. J. Reprod. Fertil., 113, 19–26.[Abstract]
Trowell, O.A. (1959) The cultures of mature organs in a synthetic medium. Exp. Cell. Res., 16, 118–147.[ISI][Medline]
Wehrenberg, U., Wulff, Ch., Husen, B. et al. (1997) The expression of SF-1/Ad4BP is related to the process of luteinization in the marmoset (Callithrix jacchus) ovary. Histochem Cell Biol., 107, 345–350.[ISI][Medline]
Wehrenberg, U., Giebel, J. and Rune, G.M. (1998) Possible involvement of transforming growth factor-ß1 and transforming growth factor-ß receptor type II during luteinization in the marmoset ovary. Tiss. Cell, 30, 360–367.
Wrana, J.L., Attisano, L., Wieser, R. et al. (1994) Mechanism of activation of the TGF-ß receptor. Nature, 370, 341–347.[Medline]
Yurchenco, P.D. and Schittny, J.C. (1990) Molecular architecture of basement membranes. FASEB J., 4, 1577–1590.[Abstract]
Submitted on December 1, 1999; accepted on March 23, 2000.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  C. Stocco, J. Kwintkiewicz, and Z. Cai Identification of regulatory elements in the Cyp19 proximal promoter in rat luteal cells J. Mol. Endocrinol., October 1, 2007; 39(4): 211 - 221. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||