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Biology of Reproduction 60, 588-593 (1999)
©Copyright 1999 Society for the Study of Reproduction, Inc.

Identification of a Nuclear Localization Signal in Activin/Inhibin ßA Subunit; Intranuclear ßA in Rat Spermatogenic Cells1

Merja Bläuer2,a, Sari Husgafvelb, Heimo Syväläc, Pentti Tuohimaaa, and Timo Ylikomia

a Molecular Endocrinology Research Unit and Graduate School of Steroid Research, Department of Anatomy, Medical School, University of Tampere, Tampere, Finland b Department of Clinical Chemistry, Tampere University Hospital, Tampere, Finland c Institute of Medical Technology, University of Tampere, FIN-33101 Tampere, Finland


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Activin is a dimeric glycoprotein hormone that was initially characterized by its ability to stimulate pituitary FSH secretion and was subsequently recognized as a growth factor with diverse biological functions in a large variety of tissues. In the testis, activin has been implicated in the auto/paracrine regulation of spermatogenesis through its cognate cell membrane receptors on Sertoli and germ cells. In this study we provide evidence for intranuclear activin/inhibin ßA subunit and show its distribution in the rat seminiferous epithelium. We have shown by transient expression in HeLa cells of ß-galactosidase fusion proteins that the ßA subunit precursor contains a functional nuclear localization signal within the lysine-rich sequence corresponding to amino acids 231-244. In all stages of the rat seminiferous epithelial cycle, an intense immunohistochemical staining of nuclear ßA was demonstrated in intermediate or type B spermatogonia or primary spermatocytes in their initial stages of the first meiotic prophase, as well as in pachytene spermatocytes and elongating spermatids primarily in stages IX–XII. In some pachytene spermatocytes, the pattern of ßA immunoreactivity was consistent with the characteristic distribution of pachytene chromosomes. In the nuclei of round spermatids, ßA immunoreactivity was less intense, and in late spermatids it was localized in the residual cytoplasm, suggesting disposal of ßA before spermatozoal maturation. Immunoblot analysis of a protein extract from isolated testicular nuclei revealed a nuclear ßA species with a molecular mass of approximately 24 kDa, which is more than 1.5 times that of the mature activin ßA subunit present in activin dimers. These results suggest that activin/inhibin ßA may elicit its biological functions through two parallel signal transduction pathways, one involving the dimeric molecule and cell surface receptors and the other an alternately processed ßA sequence acting directly within the nucleus. According to our immunohistochemical data, ßA may play a significant role in the regulation of nuclear functions during meiosis and spermiogenesis.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Spermatogenesis is the process whereby diploid spermatogonia develop to motile spermatozoa with a haploid number of chromosomes. This ordered sequence of mitotic divisions, meiosis, and cytologic differentiation is hormonally supported principally by pituitary FSH and androgens secreted by testicular Leydig cells [1]. Sertoli cells are the targets for these hormones in the seminiferous epithelium and are stimulated to produce factors that affect germ cell development and differentiation in a paracrine fashion. Conversely, spermatogenic cells have been shown to influence Sertoli cell function in vitro [2, 3]. It has become evident that within the seminiferous epithelium, spermatogenesis involves a complex network of regulatory interactions mediated by peptide hormones and growth factors [4]. Among these, a role for activins and inhibins as auto/paracrine regulators of spermatogenesis has been postulated [5]. The two hormone species were originally characterized as gonadal factors regulating the expression and release of FSH. Later, particularly activins were found to influence cell proliferation and differentiation in a wide range of tissues [6]. Activins occur as homo- or heterodimers of two closely similar ß subunits, ßA and ßB. Inhibins are heterodimers of an {alpha} subunit joined either to ßA or ßB.

Stage-specific expression of inhibin/activin subunit mRNAs has been reported in Sertoli cells during the cycle of the rat seminiferous epithelium, suggesting local regulatory functions for inhibin/activin molecules in spermatogenesis [7, 8]. It has been shown that activin stimulates the proliferation of spermatogonia in vitro [9] whereas intratesticular injection of inhibin reduces their number [10]. The regulatory effect of activin on spermatogenic cells has been further substantiated by the binding of recombinant activin to spermatogonia, pachytene spermatocytes, and spermatids in vitro [11]. Correspondingly, activin receptor II mRNAs have been localized in pachytene spermatocytes and round spermatids in isolated cells [12] and by in situ hybridization in testicular tissue sections [8, 13]. With the use of in situ hybridization, expression of the high-affinity activin receptor IIB2 was localized in spermatogonia [14]. Coexpression of the mRNAs for activin ßA and the activin-binding protein follistatin in stage IX–XI Sertoli cells [8] may be taken as a further indication of a controlled local effect of activin in spermatogenesis.

Since activin subunit mRNA expression in spermatogenic cells has not been described, available data suggest a paracrine effect of Sertoli cell activins on spermatogenesis transduced by the kinase activity of activin receptors on developing germ cells. Preliminary data suggest, however, that activin/inhibin ßA subunit may also function through direct association with the nuclei of spermatogenic cells [15, 16]. To investigate the mechanism of the intracellular distribution of activin ßA and to understand its regulatory roles during spermatogenesis, we have studied the function of the potential nuclear localization signals (NLSs) of the ßA subunit precursor and the detailed localization of immunoreactive ßA in the rat testis.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Expression Vectors and Transfection

Synthetic oligonucleotides comprising the putative NLSs corresponding to amino acids 231-244 and 277-289 of the human activin ßA subunit precursor [17] were inserted into the SmaI site in cytomegalovirus-ß-galactosidase (CMV-ß-gal) vector using conventional techniques [18]. Plasmid CMV-ß-gal was a gift from professor Pierre Chambon (IGBMC, Illkirch, France) and was constructed by Thierry Lerouge (IGBMC) by ligation of a fragment of pMC1871 (Pharmacia P-L Biochemicals, Uppsala, Sweden) containing a lacZ gene into SmaI-NheI site of pEV3S [19]. Control transfections were done with CMV-ß-gal without an NLS insert. HeLa cells were grown on glass slides in Dulbecco's Modified Eagle's Medium supplemented with 5% fetal calf serum. Transfection was performed on subconfluent cultures with a total of 5 µg DNA using the calcium phosphate coprecipitation method [20]. Two days after transfection, the cells were screened for ß-gal expression. The cells were fixed in 2% paraformaldehyde and 0.2% glutaraldehyde in PBS for 5 min on ice and left to react with 1 mg/ml X-gal (5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside; Boehringer Mannheim, Mannheim, Germany), 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, 2 mM MgCl2 in PBS for 3 h. The data are from two independent transfections.

Isolation of Cell Nuclei and Preparation of a Nuclear Protein Extract

Decapsulated testes from an adult Sprague-Dawley rat were homogenized in 8% sucrose, 25 mM KCl, 5 mM MgCl2, 20 mM tricine/NaOH, pH 7.8, using a glass-polytetrafluoroethylene homogenizer. The homogenate was filtered twice through two layers of fine nylon mesh and pelleted at 1000 x g for 10 min. The pellet was resuspended and repelleted under the same centrifugation conditions in the homogenization solution. The resulting crude nuclear fraction was resuspended in 8% sucrose solution, and a 1:2 dilution was made in 50% Nycodenz (Nycomed Pharma AS, Oslo, Norway), 25 mM KCl, 5 mM MgCl2, 20 mM tricine/NaOH, pH 7.8. Nuclei were purified by centrifuging at 15 000 x g for 1 h at 4°C on a cushion of 40% and 50% Nycodenz solutions. After centrifugation, the nuclei were collected from the interface between the 40% and 50% Nycodenz layers. Nuclear proteins were extracted by 1-h incubation in ice-cold 25 mM Tris, 1.5 mM EDTA buffer, pH 7.4, containing 2% Triton X-100 or 2% Triton X-100 together with 0.5 M NaCl.

Immunoblotting

For immunoblot analysis, the nuclear protein extracts were boiled in SDS sample buffer containing 10% mercaptoethanol. The sample was electrophoresed on a 13.5% SDS-PAGE gel and transferred to a nitrocellulose sheet. The blot was blocked in TBS (Tris-buffered saline) containing 5% BSA, incubated overnight with a polyclonal ßA-specific anti-peptide antibody (anti-ßA(88-102) [21]; 5 µg/ml), washed in TBS, and thereafter incubated with peroxidase-conjugated goat anti-rabbit IgG (Cappel, West Chester, PA) for 1 h at room temperature. After thorough washing in TBS, labeled proteins were detected by enhanced chemiluminescence as recommended by the manufacturer. The specificity of the anti-ßA antibody had been previously assessed by immunoblotting of recombinant human activin A. The antibody reacted with nonreduced activin A (apparent Mr 25 000) and the 14 000 Mr ßA monomer in reducing conditions [21].

Immunohistochemistry

Immunohistochemistry was performed on testicular tissue obtained from adult Sprague-Dawley rats and fixed in 4% paraformaldehyde, 1% CaCl2, pH 6.7. Tissue sections were stained with anti-ßA(88-102) (5 µg/ml) overnight at 4°C. Specificity of the antibody in immunohistochemistry was evaluated by testing for its ability to detect ßA immunoreactivity in human placenta previously shown to express the inhibin/activin ßA subunit [22, 23]. The staining pattern was similar to those described earlier. Preincubation of the antibody with recombinant human activin A or its corresponding peptide antigen, but not with unrelated peptides, abolished all staining in human placental tissue (unpublished results). Detection was carried out using biotin-labeled goat anti-rabbit IgG secondary antibody (Vector, Burlingame, CA) and avidin-biotin-peroxidase complex (Vector) as previously described [21, 24]. Diaminobenzidine was used as chromogen to visualize activin ßA subunit immunoreactivity.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
NLS

The efficiency of nuclear translocation by the two putative NLSs (Fig. 1) of the activin ßA subunit precursor is shown in Figure 2. The staining patterns for ß-gal activity were classified into four categories as outlined by Ylikomi et al. [25]. Preferential nuclear localization by the lysine-rich sequence 231-244 was detected, with more than 80% of the stained cells having exclusively (69.4%) or predominantly (18.2%) nuclear staining. Subcellular compartmentalization by the other putative NLS 277-289 was similar to that by CMV-ß-gal alone, with less than 13% stained cells falling into the first two categories, rendering this arginine-rich NLS motif ineffective for nuclear translocation.



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  		FIG. 1. Schematic diagram to illustrate localization of the two putative NLSs of the activin ßA precursor. For study of their potency in directing nuclear localization, they were separately fused with a cytoplasmic protein ß-gal.



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  		FIG. 2. Subcellular distribution of ß-gal activity after transfection of HeLa cells with two potential activin ßA NLSs inserted into CMV-ß-gal. The staining patterns were classified into four categories (N, N > C, N = C, N < C) according to the relative intensity of nuclear versus cytoplasmic staining. The actual numbers and percentages of cells falling into each category are indicated. For each transfection, 500 stained cells were scored.

Partial Characterization of the Nuclear Form of Activin ßA

The nuclear ßA species in testicular cells was partially characterized by SDS-PAGE and immunoblotting of a nuclear protein extract. Probing with a ßA-specific antibody directed against a 15-amino acid sequence close to the C-terminus of the ßA precursor (Fig. 1) revealed a single immunoreactive band with a molecular mass of approximately 24 kDa (Fig. 3, lane 1). The band was detectable only in the nuclear protein preparation extracted with a hypertonic (0.5 M NaCl) solution; 2% Triton X-100 used alone was ineffective in extracting the protein from the nuclei (Fig. 3, lane 2).



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  		FIG. 3. Immunoblot analysis of nuclear protein extracts from rat testicular tissue. Lane 1 represents nuclear proteins extracted with 0.5% NaCl, 2% Triton X-100; lane 2, those extracted with 2% Triton X-100. Both lanes were reacted with anti-ßA. Molecular weight markers are on the left.

Distribution of Nuclear ßA in the Rat Seminiferous Epithelium

The distribution of immunoreactive ßA in testicular cells is shown in Figure 4. In all stages of the rat spermatogenic cycle, nuclear staining was most intense in intermediate or type B spermatogonia or, as shown in Figure 4a, in spermatocytes undergoing the initial stages of the first meiotic prophase. The staining was abolished when the antibody was saturated with its corresponding peptide antigen (Fig. 4b).



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  		FIG. 4. Localization of activin ßA subunit immunoreactivity in the rat testis. a) An overview of a section stained with anti-ßA or b) anti-ßA presaturated with an excess of its peptide antigen. Seminiferous tubular stages III–V, VI–VIII, and IX–XII are represented in c, d, and e, respectively. Open arrows in a indicate immunoreactive nuclei in early primary spermatocytes. c) Open arrow indicates positive staining in intermediate or type B spermatogonia. Arrowheads point to elongated spermatids. d) Arrow points to immunoreaction in residual cytoplasm. Open arrows in e and f indicate leptotene spermatocytes. e) Arrows point to late pachytene spermatocytes and arrowheads to elongating spermatids. f) Granular appearance of ßA immunoreactivity in late pachytene spermatocytes. Bar = 50 µm (a and b); 10 µm (c, d, f); 25 µm (e).

Figure 4c shows typical nuclear immunoreactivity for ßA in elongated stage III–V spermatids. Similar staining was seen throughout stages I–V. In round spermatids, nuclear staining was less intense. In stages VI–VIII, represented by Figure 4d, ßA was detected in the residual cytoplasm. In stages IX–XII (Fig. 4, e and f), nuclear ßA was seen in leptotene/zygotene and pachytene spermatocytes as well as elongating spermatids. In some late pachytene spermatocytes, the pattern of ßA immunoreactivity was consistent with the characteristic distribution of pachytene chromosomes (Fig. 4f).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Activin ßA subunit precursor bears within its primary structure two stretches of amino acids that have resemblance to previously described functional NLSs [26] (Fig. 1). In the present study we have shown that the potential for effective nuclear import resides within the bipartite lysine-rich sequence 230-KKKKKEEEGEGKKK. The other possible NLS motif, 276-HRRRRRGLECDGK, proved ineffective on its own, yet its possible function as a proto-signal [25] functionally cooperating with the other signal to contribute to the wild-type nuclear targeting cannot be ruled out.

Location of the active NLS 52 residues upstream of the cleavage site of the mature ßA subunit implies that the nuclear ßA species might comprise a longer sequence than the mature 116-amino acid subunit (Mr 15 000) present in the 24-kDa activin and 32-kDa inhibin dimers generally acknowledged as the mature biologically active forms of these molecules. Immunoblot characterization of testicular nuclear proteins revealed that the nuclear ßA species has a molecular mass of 24 kDa. Solubilization of both membranes of the nuclear envelope with 2% Triton X-100 was ineffective in extracting the immunoreactive protein. Instead, a hypertonic solution was required, indicating strong association of the protein with nuclear structures [27].

Mapping of the ßA amino acid sequence upstream of the active NLS revealed a putative endoproteolytic cleavage site 185-RK representing a classical dibasic site requisite for posttranslational processing of many prohormones to biologically active peptides by endoproteolytic enzymes [28]. Cleavage of the ßA precursor at this site would produce a polypeptide sequence having its deduced molecular weight in exact agreement with the detected molecular mass of the nuclear polypeptide. Testicular tissue contains elevated quantities of many endoproteases (some of which are testis-specific), which are implicated in the processing of the peptides and proteins that mediate interactions between testicular cells [29–31]. Our earlier observations suggest that differentially processed inhibin {alpha} precursors may be involved in spermatogonial differentiation and maintenance of spermatogenesis in the hamster testis [32]. It has also been shown that aside from the 32-kDa inhibin dimer, biologically active, high molecular weight forms of inhibins composed of differentially processed subunit precursors can be found in the peripheral circulation [33–37]. Species differences in inhibin precursor processing have been proposed [33], and it is reasonable to assume that tissue differences might also exist.

In its ability for nuclear localization, activin ßA appears unique among the activin/inhibin-related proteins. Whereas nuclear ßA has been described in addition to testicular tissue [15, 16] also in the central nervous system [38], nuclear staining of {alpha} and ßB subunits has not been reported, and in their amino acid sequence they lack any recognizable NLS. The same also holds for the predicted sequences of the newly found activin ß subunits ßC, ßD, and ßE [39–41]. It is clear from our present study and previous reports on ßA expression in testicular tissue [8, 42] that, as for the activin/inhibin family, ßA has a distinctive role in the regulation of spermatogenesis. It is possible that some of the pleiotropic effects reported for activin also outside the testis and the brain, including its functions in embryonic development, could be mediated by direct nuclear contribution.

Our present immunohistochemical data show that in all stages of the rat seminiferous epithelial cycle, nuclear ßA is most prominent in spermatogonia or primary spermatocytes undergoing the initial stages of the first meiotic prophase. In stages IX–XII, ßA immunoreactivity was also detected in the nuclei of pachytene spermatocytes and elongated spermatids. Since expression of ßA mRNA has been detected solely in Sertoli cells [8], it may be assumed that the protein is taken up by the germ cells after translation in Sertoli cell cytoplasm. Another possibility is that germ cells have their own ßA production in an amount not detectable by in situ hybridization. In this case, ßA might be acting as an intracrine regulator within its cell of origin [43]. Either condition presupposes specific mechanisms for the secretion and processing of ßA in testicular cells. Available data indicate that in other families of polypeptide hormones and growth factors where nuclear localization is not uncommon [27], nuclear entrance can be accomplished by various means, including the competing effects of signals for secretion and nuclear localization within the same protein [44].

Interestingly, ßA seems to be disposed with the residual cytoplasm before the spermatozoal stage. Even though this ßA would be taken up by Sertoli cells with the rest of the residual cytoplasm, no immunoreactivity could be demonstrated in Sertoli cell cytoplasm in this study. This is compatible with previous descriptions of sparse ßA immunoreactivity in Sertoli cells [16] and leaves the ultimate fate of the nuclear ßA obscure. In accordance with the findings of Shaha et al. [16], who described nuclear ßA exclusively in zygotene and pachytene spermatocytes, we found ßA distributed in a pattern similar to that for pachytene chromosomes in stage IX–XII spermatocytes. The suggested regulatory role of activin in the meiotic nuclei of germ cells gains further emphasis from the studies of Kogawa et al. [45] and Ogawa et al. [46], who showed the presence of immunoreactive follistatin in the nuclei of pachytene spermatocytes and round spermatids. Exact colocalization of ßA and follistatin mRNAs in stage IX–XI Sertoli cells [8] gives additional support to the proposed view of a close interplay between the two molecules in the rat seminiferous epithelium. The nature of their possible functional relationship within the nucleus remains to be determined.

In summary, activin/inhibin ßA may be added to the growing list of polypeptide hormones and growth factors having a potential for direct intranuclear function [47–50]. The present data suggest that within the testis, ßA might play a role in two parallel signal transduction pathways: one involving the dimeric molecule and cell surface receptors, and the other an alternately processed ßA sequence acting directly within the nucleus.


   ACKNOWLEDGMENTS
 
We wish to thank Ms. Hilkka Mäkinen for skilled technical assistance. Professor Pierre Chambon and Thierry LeRouge from IGBMC, Illkirch, France, are gratefully acknowledged for donating the plasmid CMV-ß-gal vector.


   FOOTNOTES
 
1 This work was supported by grants from the Medical Research Fund of Tampere University Hospital and the University of Tampere. Back

2 Correspondence: Merja Bläuer, Department of Anatomy, Medical School, University of Tampere, P.O. Box 607, FIN-33101 Tampere, Finland. FAX: 358 3 2156170; blauer{at}csc.fi Back

Accepted: October 6, 1998.

Received: February 5, 1998.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Steinberger E. Hormonal control of mammalian spermatogenesis. Physiol Rev 1971; 51:1–22.[Medline]
  2. Djakiew D, Dym M. Pachytene spermatocyte proteins influence Sertoli cell function. Biol Reprod 1988; 39:1193–1205.[Abstract]
  3. Allenby G, Foster PMD, Sharpe RM. Evidence that secretion from the adult rat testis is regulated by specific germ cell types: correlation between in vivo and in vitro studies. Endocrinology 1991; 128:467–476.[Abstract/Free Full Text]
  4. Spiteri-Grech J, Nieschlag E. Paracrine factors relevant to the regulation of spermatogenesis—a review. J Reprod Fertil 1993; 98:1–14.[Abstract/Free Full Text]
  5. Mather JP, Woodruff TK, Krummen LA. Paracrine regulation of reproductive function by inhibin and activin. Proc Soc Exp Biol Med 1992; 201:1–15.[CrossRef][Medline]
  6. Vale W, Hsueh A, Rivier C, Yu J. The inhibin/activin family of hormones and growth factors. In: Sporn MB, Roberts AB (eds.), Peptide Growth Factors and Their Receptors II. Handbook of Experimental Pharmacology, vol. 95. Berlin: Springer Verlag; 1990: 211–248.
  7. Bhasin S, Krummen LA, Swerdloff RS, Morelos BS, Kim WH, DiZerega GS, Ling N, Esch F, Shimasaki S, Toppari J. Stage dependent expression of inhibin {alpha} and ß-B subunits during the cycle of the rat seminiferous epithelium. Endocrinology 1989; 124:987–991.[Abstract/Free Full Text]
  8. Kaipia A, Penttilä T-L, Shimasaki S, Ling N, Parvinen M, Toppari J. Expression of inhibin ßA and ßB, follistatin and activin-A receptor messenger ribonucleic acids in the rat seminiferous epithelium. Endocrinology 1992; 131:2703–2710.[Abstract/Free Full Text]
  9. Mather J, Attie K, Woodruff T, Rice G, Phillips D. Activin stimulates spermatogonial proliferation in germ-Sertoli cell cocultures from immature rat testis. Endocrinology 1990; 127:3206–3214.[Abstract/Free Full Text]
  10. van Dissel-Emiliani F, Grootenhuis A, de Jong F, de Rooij D. Inhibin reduces spermatogonial numbers in testes of adult mice and Chinese hamsters. Endocrinology 1989; 125:1899–1903.[Medline]
  11. Woodruff TK, Borree J, Attie KM, Cox ET, Rice GC, Mather JP. Stage-specific binding of inhibin and activin to subpopulations of rat germ cells. Endocrinology 1992; 130:871–881.[Abstract/Free Full Text]
  12. de Winter JP, Themmen APN, Hoogerbrugge JW, Klaij IA, Grootegoed JA, De Jong FH. Activin receptor mRNA expression in rat testicular cell types. Mol Cell Endocrinol 1992; 83:R1-R8.
  13. Cameron VA, Nishimura E, Mathews LS, Lewis KA, Sawchenko PA, Vale WW. Hybridization histochemical localization of activin receptor subtypes in rat brain, pituitary, ovary, and testis. Endocrinology 1994; 134:799–808.[Abstract/Free Full Text]
  14. Kaipia A, Parvinen M, Toppari J. Localization of activin receptor (ActR-IIB2) mRNA in the rat seminiferous epithelium. Endocrinology 1993; 132:477–479.[Abstract/Free Full Text]
  15. Bläuer M, Vilja P, Hjerppe T, Tuohimaa P. Localization of inhibin {alpha}- and ßA-subunits in the rat testis. In: Program of the 6th European workshop on molecular and cellular endocrinology of the testis; 1990; Mariehamn, Finland. Abstract D9.
  16. Shaha C, Morris PL, Chen C-LC, Vale W, Bardin W. Immunostainable inhibin subunits are in multiple types of testicular cells. Endocrinology 1989; 125:1941–1950.[Abstract/Free Full Text]
  17. Mason AJ, Niall HD, Seeburg PH. Structure of two human ovarian inhibins. Biochem Biophys Res Commun 1986; 135:957–964.[CrossRef][Medline]
  18. Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning: A Laboratory Manual, 2nd ed. New York: Cold Spring Harbor; 1989.
  19. Matthias P, Müller MM, Schreiber E, Rusconi S, Schaffner W. Eucaryotic expression vectors for the analysis of mutant proteins. Nucleic Acid Res 1989; 17:6418.[Free Full Text]
  20. Wigler M, Pellicer A, Silverstein S, Axel R. Biochemical transfer of single-copy eucaryotic genes using total cellular DNA as donor. Cell 1978; 14:725–731.[CrossRef][Medline]
  21. Bläuer M, Kohonen J, Leivonen I, Vilja P, Tuohimaa P. Inhibin {alpha}- and ßA-subunit immunoreactivity in the chicken embryo during morphogenesis. Development 1992; 115:1079–1086.[Abstract]
  22. Minami S, Yamoto M, Nakano R. Immunohistochemical localization of inhibin/activin subunits in human placenta. Obstet Gynecol 1992; 80:410–414.[Medline]
  23. Rabinovici J, Goldsmith PC, Librach CL, Jaffe RB. Localization and regulation of the activin-A dimer in human placental cells. J Clin Endocrinol Metab 1992; 75:571–576.[Abstract]
  24. Bläuer M, Tuohimaa P. Activin ßA- and ßB-subunit expression in the developing chicken bursa of Fabricius. Endocrinology 1995; 136:1482–1487.[Abstract]
  25. Ylikomi T, Bocquel MT, Berry M, Gronemeyer H, Chambon P. Cooperation of proto-signals for nuclear accumulation of estrogen and progesterone receptors. EMBO J 1992; 11:3682–3694.
  26. Garcia-Bustos J, Heitman J, Hall MN. Nuclear protein localization. Biochim Biophys Acta 1991; 1071:83–101.[Medline]
  27. Burwen SJ, Jones AL. The association of polypeptide hormones and growth factors with the nuclei of target cells. TIBS 1987; 12:159–162.
  28. Doherty K, Steiner DF. Post-translational proteolysis in polypeptide hormone biosynthesis. Annu Rev Physiol 1982; 44:625–638.[CrossRef][Medline]
  29. Torii S, Yamagishi T, Murakami K, Nakayama K. Localization of Kex2-like processing endoproteases, furin and PC4, within the mouse testis by in situ hybridization. FEBS Lett 1993; 316:12–16.[CrossRef][Medline]
  30. Kuo W-L, Montag AG, Rich Rosner M. Insulin-degrading enzyme is differentially expressed and developmentally regulated in various rat tissues. Endocrinology 1993; 132:604–611.[Abstract/Free Full Text]
  31. Chesneau V, Pierotti AR, Barré N, Créminon C, Tougard C, Cohen P. Isolation and characterization of a dibasic selective metalloendopeptidase from rat testes that cleaves at the amino terminus of arginine residues. J Biol Chem 1994; 269:2056–2061.[Abstract/Free Full Text]
  32. Tuohimaa P, Bläuer M, Bergmann M, Aumüller G. Changes in location and processing of inhibin {alpha}-subunit precursors during sexual maturation of the Djungarian hamster testis. Endocrinology 1993; 132:629–633.[Abstract/Free Full Text]
  33. Sugino K, Nakamura T, Takio K, Miyamoto K, Hasagawa Y, Igarashi M, Titani K, Sugino H. Purification and characterization of high molecular weight forms of inhibin from bovine follicular fluid. Endocrinology 1992; 130:789–796.[Abstract/Free Full Text]
  34. Robertson DM, Sullivan J, Watson M, Cahir N. Inhibin forms in human plasma. J Endocrinol 1995; 144:261–269.[Abstract/Free Full Text]
  35. Good TEM, Weber PSD, Ireland JLH, Pulaski J, Padmanabhan V, Schneyer AL, Lambert-Messerlian G, Ghosh BR, Miller WL, Groome N, Ireland JJ. Isolation of nine different biologically and immunologically active molecular variants of bovine follicular inhibin. Biol Reprod 1995; 53:1478–1488.[Abstract]
  36. Mason AJ, Farnworth PG, Sullivan J. Characterization and determination of the biological activities of noncleavable high molecular weight forms of inhibin A and activin A. Mol Endocrinol 1996; 10:1055–1065.[Abstract/Free Full Text]
  37. Robertson DM, Cahir N, Findlay JK, Burger HG, Groome N. The biological and immunological characterization of inhibin A and B forms in human follicular fluid and plasma. J Clin Endocrinol Metab 1997; 82:889–896.[Abstract/Free Full Text]
  38. Sawchenko PE, Plotsky PM, Pfeiffer SW, Cunningham ET, Vaughan J, Rivier J, Vale W. Inhibin ß in central neural pathways involved in the control of oxytocin secretion. Nature 1988; 334:615–617.[CrossRef][Medline]
  39. Hötten G, Neidhardt H, Schneider C, Pohl J. Cloning of a new member of the TGF-ß family: a putative new activin ßC chain. Biochem Biophys Res Commun 1995; 206:608–613.[CrossRef][Medline]
  40. Oda S, Nishimatsu S, Murakami K, Ueno N. Molecular cloning and functional analysis of a new activin ß subunit: a dorsal mesoderm-inducing activity in Xenopus. Biochem Biophys Res Commun 1995; 210:581–588.[CrossRef][Medline]
  41. Fang J, Yin W, Smiley E, Wang SQ, Bonadio J. Molecular cloning of the mouse activin ßE subunit gene. Biochem Biophys Res Commun 1996; 228:669–674.[CrossRef][Medline]
  42. Meunier H, Rivier C, Evans RM, Vale W. Gonadal and extragonadal expression of inhibin {alpha}, ßA and ßB subunits in various tissues predicts diverse functions. Proc Natl Acad Sci 1988; 85:247–225.[Abstract/Free Full Text]
  43. Logan A. Intracrine regulation at the nucleus—a further mechanism of growth factor activity. J Endocrinol 1990; 125:339–343.[Abstract/Free Full Text]
  44. Kiefer P, Acland P, Pappin D, Peters G, Dickson C. Competition between nuclear localization and secretory signals determines the subcellular fate of a single CUG-initiated form of FGF3. EMBO J 1994; 13:4126–4136.[Medline]
  45. Kogawa K, Ogawa K, Hayashi Y, Nakamura T, Titani K, Sugino H. Immunohistochemical localization of follistatin in rat tissues. Endocrinol Jpn 1991; 38:383–391.[Medline]
  46. Ogawa K, Hashimoto O, Kurohmaru M, Mizutani T, Sugino H, Hayashi Y. Follistatin-like immunoreactivity in the cytoplasm and nucleus of spermatogenic cells in the rat. Eur J Endocrinol 1997; 137:523–529.[Abstract]
  47. Henderson J. Nuclear targeting of secretory proteins. Mol Cell Endocrinol 1997; 129:1–5.[CrossRef][Medline]
  48. Moroianu J, Riordan JF. Nuclear translocation of angiogenic proteins in endothelial cells: an essential step in angiogenesis. Biochemistry 1994; 33:12535–12539.[CrossRef][Medline]
  49. Steward R. Relocalization of the dorsal protein from the cytoplasm to the nucleus correlates with its function. Cell 1989; 59:1179–1188.[CrossRef][Medline]
  50. Shiurba RA, Jing N, Sakakura T, Godsave SF. Nuclear translocation of fibroblast growth factor during Xenopus mesoderm induction. Development 1991; 113:487–493.[Abstract]



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