0013-7227/92/1306-3459$03.00/O Endocrinology

Copyright

0 1992 by The Endocrine

Vol. 130, No. 6 Printed in U.S.A.

Society

Expression of Transforming Growth Factor-P Isoforms (p2 and ,63) in the Mouse Uterus: Analysis of the Periimplantation Period and Effects of Ovarian Steroids* SANJOY K. DAS, KATHLEEN AND SUDHANSU K. DEY

C. FLANDERS,

GLEN

K. ANDREWS,

and Molecular Departments of Obstetrics-Gynecology and Physiology (Sa.K.D., S.K.D.) and Biochemistry Biology (G.K.A.), University of Kansas Medical Center, Ralph L. Smith Research Center, Kansas City, Kansas 66160-7338; and the Laboratory of Chemopreuention, National Cancer Institute, National Institutes of Health (K.C.F.), Bethesda, Maryland 20892

in total uterine poly(A)+ RNA on Dl-D6. This transcript was detected in myometrial RNA samples on D7 and D8 of pregnancy or D8 of pseudopregnancy, but was not detected in RNA from the deciduum on D7 and D8 or in that from deciduomata on D8. The effects of ovarian steroids on TGFp2 and TGFP3 mRNAs were examined in uteri of adult ovariectomized mice. Uterine TGF/32 or TGFP3 mRNA persisted in ovariectomized mice. However, an injection of E, induced a rapid (6 h), but transient, induction (-3. to 4-fold) of TGFP2 mRNA. An injection of P, had no effect on TGF/32 mRNA levels, and coinjection of P, with E, did not antagonize the E2-stimulated transient accumulation of TGFP2 mRNA. In comparison, neither an injection of E, nor one of P, exerted significant effects on TGFP3 mRNA levels. The results of this study establish that 1) TGFp2 and TGF/33 genes are expressed in the periimplantation uterus; 2) epithelial, myometrial, and decidual cells are primary sites of TGFP2 synthesis, whereas myometrial cells are the primary site of synthesis of TGF@3 during the periimplantation period, and 3) E, may play a role in the regulation of TGFP2 mRNA levels, but an acute treatment with P4 and/or E, does not regulate TGF/33 gene expression. The distinct uterine cell type-specific expression of TGF@Z and TGFP3 suggests that these growth factors may have different functional roles during the periimplantation period. (Endocrinology 130: 3459-3466, 1992)

ABSTRACT. Expression of P-type transforming growth factor genes (TGFPP and TGFp3) in the mouse uterus during the periimplantation period and in response to an acute exposure to 17/3-estradiol (EJ and progesterone (P,) was studied using Northern blot hybridization and/or immunocytochemistry. Polyclonal antipeptide antibodies specific for TGFp2 or TGFP3 were employed for immunocytochemistry. In the preimplantation uterus [days (D) 1-4 of pregnancy; day 1 = vaginal plug], immunostaining for TGFP2 was observed in luminal and glandular epithelia as well as in myometrium and vascular smooth muscle. In the postimplantation period (D5-D8), TGFP2 immunostaining was also detected in decidual cells. In contrast, TGF/33 immunostaining was restricted to the myometrium and vascular smooth muscle throughout the periimplantation period (Dl-D8). Antisense TGFP2 and TGFP3 RNA probes were employed for Northern blotting. Northern blot hybridization revealed four TGF/32 transcripts (-6.0, 5.0, 4.0, and 3.5 kilobases) in total uterine poly(A)’ RNA on Dl-D6 and in poly(A)’ RNA from the deciduum and myometrium collected on D7 and D8 of pregnancy. These TGF@2 transcripts were also detected in isolated samples of deciduomata or myometrium obtained from D8 pseudopregnant mice in which the decidual cell reaction was induced experimentally on D4. The levels of these transcripts remained relatively constant during the periimplantation period. Northern blot analysis detected a 3.8-kilobase TGFP3 transcript

T

HE PROCESSES that lead to synchronized development of the preimplantation embryo into the blastocyst and preparation of the uterus to the receptive state are central to successful implantation. Coordination and maintenance of these events are mediated by

ovarian estrogen and progesterone (1). However, the mechanisms by which these ovarian steroids direct these processesare not clearly understood. The documentation that several polypeptide growth factors and their receptors are expressed in the uterus and embryo during early pregnancy suggests that at least some of the effects of these ovarian steroids in the processes of implantation are mediated by autocrine/paracrine effects of growth factors (2-15). The P-type transforming growth factors (TGFP), a family of structurally homologous dimeric proteins, have been suggested to play important roles in embryogenesis

Received December 17, 1991. Address all correspondence and requests for reprints to: S. K. Dey, Departments of Obstetrics/Gynecology and Physiology, University of Kansas Medical Center, MRRC 37/317, 39th and Rainbow Boulevard, Kansas City, Kansas 66160-7338. *This work was supported in part by grants from the NICHHD (HD-12304 to S.K.D.), the NIEHS (ES-04725 to G.K.A.), and the Wesley Foundation, Wichita, KS (to S.K.D. and G.K.A.).

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(16-19). Five isoforms of TGFP (TGFPs l-5) have been identified, of which TGF@s l-3 are prevalent in mammals (20-23). These three proteins are synthesized as prepropeptides. After secretion they are cleaved to produce peptides of 112-114 amino acids that dimerize to give rise to active growth factors (24-26). Although TGFPs l-3 are 70-80% identical in the mature regions, they share only 27% sequence identity in their precursor regions (27). These TGFPs exert diverse biological functions, including regulation of proliferation, tissue remodelling, extracellular matrix formation, control of cell surface molecules, and immunomodulation (28-30). Because embryo-uterine interactions during the process of implantation involve each of these processes, these growth factors may have important roles during the periimplantation period. Although TGFp isoforms manifest overlapping biological functions, their relative potencies may vary, and there is limited evidence for their distinct biological effects (19, 31, 32). Therefore, information on cell type-specific expression of TGFP isoforms is necessary to better understand their potentially unique functions. To date, expression of TGFPl has been examined in the mouse uterus during the periimplantation period (12). The present investigation was undertaken to examine the cell type-specific expression and regulation of TGFp2 and TGFP3 in the mouse uterus.

Materials

and Methods

Animals CD-1 mice (Charles River Laboratories, Raleigh, NC) were housed in the animal care facility at the University of Kansas Medical Center in accordance with NIH standards for the care and use of experimental animals. Female mice (48 days old) were mated with fertile males of the same strain. The morning on which a vaginal plug was found was considered day 1 of pregnancy (Dl). Animals were killed between 0830-0930 h on the indicated day of pregnancy (Dl-D8). On Dl-6, whole uteri were collected, whereas on D7 and D8, the deciduum and myometrium were separated. Pseudopregnant mice, produced by mating with vasectomized males, were used for induction of deciduomata as follows. On D4 of pseudopregnancy, 50 ~1 sesame seed oil were injected intraluminally to initiate the process of decidualization. Mice were killed on D8, and deciduomata and myometria were separated. Animals in steroid treatment groups were bilaterally ovariectomized and allowed to recover for at least 7 days. Ovariectomized mice were then given a single sc injection of sesame oil (0.1 ml/mouse), 17pestradiol (E,; 250 rig/mouse), progesterone (P4; 2 mg/mouse), or a combination of the same doses of Ez and P4 dissolved in sesame oil. In a separate experiment, ovariectomized mice were injected with P, for 3 days, and on the fourth day they either received another injection of Pq or a combination of Pq and Ez. The mice were killed 6, 12, and 24 h after injection. Whole uteri or isolated uterine tissue preparation were processed for

Endo. 1992 Voll30. No 6

immunohistochemistry or extraction of RNA. Each experiment was repeated at least twice. Antibody Rabbit polyclonal antibodies to TGFP2 and TGFP3 were produced using synthetic peptides as immunogens. Antibodies were directed against amino acids 50-75 of mature TGFP2 (anti-P 50-75) and amino acids 81-100 of the prepro region of TGFP3 (anti-P 81-100) (33, 34). The characterization and specificity of these antibodies have been described previously (33-35). Immunohistochemistry Immunolocalization was performed as described previously (4, 12). Uteri were excised, cleaned of fat, cut into 4- to 6-mm pieces, and fixed in Bouin’s solution for 24 h. Paraffin-embedded tissues were sectioned at 7 pm and mounted onto polyL-lysine-coated slides. Sections were deparaffinized, hydrated in PBS for 20 min, and then incubated in 10% normal goat serum (blocking solution) for 10 min. Sections were then incubated for 22 h at 4 C in primary antibodies to TGFPP (1 pg/ ml) or TGF/33 (1.25 pg/ml) diluted in PBS. Immunostaining was performed using a Zymed-Histostain-SP kit for rabbit primary antibody (Zymed Laboratories, San Francisco, CA). This kit used a biotinylated secondary antibody, a horseradish peroxidase-streptavidin conjugate, and a substrate chromogen mixture (36). Blocking of endogenous peroxidase activity was achieved by a 35-set incubation in 0.23% periodic acid in PBS after incubation of sections with secondary antibody. After immunostaining, sections were stained lightly with hematoxylin, mounted, and examined under a brightfield microscope. Control experiments included incubation of the sections with primary antibodies neutralized with a lo-fold molar excess of immunizing peptides. Red deposits indicated the sites of immunoreactive protein. Isolation

of

RNA

Total RNA was extracted from uteri and decidual tissues using a modification of a procedure described by Han et al. (37). Tissues (1 g) representing several animals per group were homogenized in 20 ml 5 M guanidine thiocyanate (Fluka, Ronkonkoma, NY), 25 mM EDTA, 50 mM Tris (pH 7.4), and 8% ,&mercaptoethanol, and nucleic acids were precipitated with 0.75 vol cold ethanol. The precipitate was rapidly dissolved by homogenization in 6 M guanidine hydrochloride, 25 mM EDTA, and 10 mM P-mercaptoethanol, and RNA was precipitated for 3 h at -20 C by the addition of 0.5 vol cold ethanol and 0.05 ~011 M acetic acid. This procedure was repeated twice, and the final pellet was dissolved in alkaline buffer (75 mM NaCl, 25 mM EDTA, and 0.5% sodium dodecyl sulfate), extracted with phenol-chloroform, and adjusted to 0.3 M ammonium acetate, and RNA was precipitated with 2.5 vol ethanol at -20 C for 3 h. The precipitate was washed in 70% ethanol and dissolved in water, and RNA was quantitated by measuring the absorbance at 260 nm. Polyadenylated RNA [poly(A)+] was isolated from whole uterine RNA by oligo(dt)-cellulose column chromatography (38). Yields of poly(A)+ RNA were similar among the uterine samples (-2.5% of the total RNA), and at least two

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TGF/32 independent preparations each experiment.

of poly(A)+

AND

RNA were analyzed

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Hybridization probes cDNA clones for mouse TGFP2 and TGFP3 were provided by Dr. Harold Moses (Vanderbilt University School of Medicine, Nashville, TN). The TGFP2 cDNA (nucleotides 15111953) corresponds to the N-terminal glycopeptide-coding region of TGFP2 mRNA (39). The TGFP3 cDNA (nucleotides sequence 831-1440) corresponds to the N-terminal glycopeptide-coding region of TGF/33 mRNA (40). A cDNA clone for mouse lactoferrin (LF) (41) was provided by Dr. Tina Teng (NIEHS, Research Triangle Park, NC). cDNA clones were subcloned into plasmid vectors containing a promoter for Sp6 RNA polymerase and were used as templates for the Sp6directed synthesis of 3ZP-labeled cRNA probes. Probes had specific activities of about 2 x 10’ dpm/pg.

Northern blot hybridization Poly(A)’ RNA (2.0 pg) was denatured, separated by formaldehyde-agarose gel electrophoresis, and transferred to nylon membranes. RNA was cross-linked to the membranes by UV irradiation (Spectrolinker, XL-1500, Spectronics Corp., Westbury, NY) for 12 sec. Northern blots were prehybridized, hybridized, and washed, as described previously (42, 43). Hybridization was carried out under highly stringent conditions at 68 C in the presence of 40% formamide, 3 x SET (1 x SET = 150 mM NaCl, 5 mM EDTA, and 10 mM Tris/HCl, pH 8.0), and 10% dextran sulfate (12). Blots were rehybridized to different probes. Stripping of hybridized probe before subsequent rehybridization was achieved by boiling in 0.05 x SSC and 0.1% sodium dodecyl sulfate for 3 min. After each hybridization, the blots were subjected to autoradiography and radioimage quantitation, using the radioanalytic image system (Ambis Systems, Inc., San Diego, CA).

Results Immunocytochemistry of TGF/32 and TGFp3 in the periimplantation uterus The cell type-specific

accumulation

of immunoreactive

TGFP2 and TGFP3 was examined by immunocytochemistry. Red deposits in tissue sections indicated the sites of immunoreactivity. Control sections incubated with primary antibodies preneutralized with an excess of specific antigen showed greatly reduced or no immunostaining (data not shown). On Dl-D4 (preimplantation period), immunostaining for TGFP2 was primarily limited to the luminal and glandular epithelia and the myometrium (Fig. 1). However, the immunostaining on D3 and D4 was less intense than that on Dl and D2. On D&D8 (postimplantation period), in addition to the epithelial and myometrial localization, the decidua exhibited immunostaining (Fig. 1). Immunoreactive TGFp2 was also detected in the embryo during the postimplantation period. .In contrast, immunostaining for TGFP3

FIG. 1. Immunocytochemical localization of TGFP2 in the mouse uterus on Dl-D8 of pregnancy. Uteri were collected on the indicated day of pregnancy, and Bouin’s-fixed paraffin-embedded sections (7 pm) were mounted onto poly-L-lysine coated slides. Each slide contained uterine sections from Dl-D4 or D5-D8 of pregnancy. After deparaffinization and hydration, sections were incubated with primary antibody. Immunostaining employed the avidin-biotin-peroxidase complex technique, in which red deposits indicate positive immunostaining. Controls were performed to ensure that endogenous peroxidase was blocked. Sections showed greatly reduced or no immunostaining after incubation with the primary antibody preneutralized with an excess of the antigenie peptide (data not shown). The top row from left to right represents photomicrographs of uterine sections on Dl-D3, respectively. The middle ro’owfrom left to right represents those on D4-D6, respectively. The photomicrograph in the bottom row at the center represents a uterine section on D8 of pregnancy. Photomicrographs of Dl-D4 uterine sections were at Xl00 magnification, and those of D5, D6, and D8 were at x40 magnification. Although not shown here, the immunostaining pattern for TGFP2 in D7 uterine sections was similar to that in D6 or D8 sections. LE, Luminal epithelium; GE, glandular epithelium; CM, circular muscle; LM, longitudinal muscle, E, embryo; PDZ, primary decidual zone; SDZ, secondary decidual zone.

on all days examined (Dl-D8) was restricted to the myometrium (Fig. 2). Immunostaining in the longitudinal muscle cells was more intense than that in the circular muscle cells. In addition, vascular smooth muscle cells also showed immunostaining for TGF/32 and TGFP3. TGFp2 and TGF/33 mRNAs in the periimplantation uterus Relative levels of TGFp2 and TGFP3 mRNAs in the uterus during the periimplantation period were examined

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ata, isolated from D8 pseudopregnant mice (Fig. 3, bottom right panel). The data suggest that TGFP3 mRNA is abundant in the myometrium. This mRNA could be readily detected in total uterine RNA (data not shown). Furthermore, TGFP3 mRNA is apparently more abundant than TGFP2 mRNA in total uterine poly(A)+ RNA based on comparison of hybridization signals (Figs. 3 and 4). Effects of Ez and/or P4 on uterine TGFp2 and TGF/33 mRNAs

FIG. 2. Immunocytochemical localization of TGFP3 in the mouse uterus on Dl-D8 of pregnancy. Immunolocalization of TGFP3 was performed as described in Fig. 1.

by Northern blot hybridization using 32P-labeled cRNA probes. In these experiments, mouse lung and placental poly(A)+ RNAs served as positive controls, while that from the spleen served as a negative control for TGFP2 and TGFP3 mRNAs (44). Four TGF/32 transcripts [-6.0, 5.0,4.0, and 3.5 kilobases (kb)] were detected in the lung, placenta, and uterus on Dl-D8 of pregnancy (Fig. 3, top left and right panels). Steady state levels of these transcripts remained virtually unaltered during this period. However, the 6.0- and 4.0-kb transcripts were either absent or present in low abundance in D8 deciduum compared to levels in the D8 myometrium or whole uterus. In contrast, all four transcripts were present in both decidual and myometrial tissues obtained from D8 pseudopregnant mice in which the decidual reaction was induced experimentally on D4 (Fig. 3, top right panel). A single 3.8-kb TGF/33 transcript was detected in poly(A)+ RNA from intact uteri on Dl-D6, but when poly(A)+ RNA was extracted from the surgically separated myometrium and deciduum on D7 and D8, TGFP3 mRNA was detected only in the myometrial samples (Fig. 3, bottom left panel). TGF/33 mRNA was also detected in myometrial RNA, but not in that of deciduom-

The effects of an acute exposure to ovarian steroids on uterine TGFP2 and -@3 gene expression were examined in adult ovariectomized mice by Northern blot hybridization and quantitation of the amount of probes hybridized, as determined by radioimage analysis of the filters (Figs. 4 and 5) using an Ambis system (Ambis Systems, Inc., San Diego, CA). Uterine TGFP2 and $33 mRNAs persisted in ovariectomized mice, whereas LF mRNA levels were dramatically reduced (Fig. 4, compare ovariectomized us. D2). However, a transient 3- to 4-fold induction of two TGFP2 transcripts (6.0 and 4.0 kb) was detected 6 h after an injection of EZ, and these mRNAs declined to basal levels by 12 h (Figs. 4 and 5). In contrast, an injection of Es had little effect on TGFP3 mRNA levels, but dramatically induced LF mRNA, which was elevated to 14-fold at 24 h (Figs. 4 and 5). An injection of P4 had little effect on uterine TGFPB, TGFP3, or LF mRNA levels. A coinjection of Pq did not antagonize the E2-induced transient accumulation of TGFP2 mRNA, but completely prevented the effects of E2 on LF gene expression, as reported previously (41). In contrast, while P4 priming for 4 days had little effect on uterine TGFP2 mRNA constitutive levels, this treatment abolished Ez-induced transient accumulation of TGFP2 mRNA (data not shown). The same blots were hybridized to TGFP2, TGFP3, and LF probes, which provided an internal control to validate conclusions regarding the differential effects of Ez/P4 on the expression of these genes. Overall, these experiments suggest that ovarian steroids exert a modest influence on uterine TGFP2 and $3 gene expression.

Discussion Previously reported studies from this laboratory (12) in conjunction with the present study demonstrate that all three mammalian TGFP isoforms (TGFPs l-3) are expressed in the periimplantation mouse uterus in a cell type-specific manner. Expression of TGF/31 is primarily limited to the luminal and glandular epithelia during the preimplantation period (Dl-D4) and to the deciduum during the postimplantation period (D5-D8) (12). The patterns of TGF/32 gene expression are similar to those

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Kb

- 28s TGF-

3.8-

I&

- 18s

Day

of

Pregnancy

FIG. 3. Northern blot detection of TGFP2 and TGFP3 mRNAs in the mouse uterus during the periimplantation period (Dl-D8). Whole uterine poly(A)+ RNA from Dl-D6 or deciduum (Dee) and myometrium (Myo) on D7 and D8 of pregnancy was analyzed (left panels). Poly(A)+ RNA was also obtained from deciduomata (DM) and myometrium (Myo) on D8 of pseudopregnancy (right panels). Deciduomata was induced experimentally by injection of 50 ~1 sesame oil into the uterine lumen on D4 of pseudopregnancy. Poly(A)+ RNA (2.0 rg) was separated by formaldehyde-agarose gel electrophoresis, transferred to nylon membrane, and hybridized to 32P-labeled RNA probes complementary to mouse TGFP2 and TGFP3 mRNAs. RNA samples in duplicate gels were stained with acridine orange, and the mobilities of the small amounts of 2% and 1% ribosomal RNAs remaining in the poly(A)+ RNA samples are indicated. Four major TGFp2 transcripts (approximate sizes of 6.0, 5.0, 4.0, and 3.5 kb) and a single 3.8-kb TGFP3 transcript were detected. Poly(A)+ RNA samples from mouse lung (Lu) and placenta (Pl) served as positive controls, and those from spleen (Sp) served as a negative control (44).

of TGFPl, except that the TGFP2 gene is also expressed in the uterine myometrium. However, the TGFP3 gene is expressed exclusively in the myometrium. The significance of multiple TGFP2 transcripts in the uterus is not yet clear. Analyses of cDNA have led to the suggestion that this heterogeneity might reflect the presence of multiple polyadenylation signals in the 3’-untranslated sequence (45). It has been reported that heterogeneity of TGF/31 transcripts is due to the presence of different transcriptional initiation sites (45). However, differential splicing of the TGF/32 mRNA could also be involved. There is evidence that the insertion of an additional exon occurs in one of the TGFP2 mRNA species, and this may result in synthesis of a larger TGFP2 precursor segment (46). The absence of 6.0- and 4.0-kb TGFP2 transcripts in decidua, but not in deciduomata, suggests that the presence of the embryo may influence the levels of these transcripts. The differential expression of TGFP isoforms in various uterine cell types, each endowed with specific functions during the periimplantation period, suggests that in addition to overlapping functions (47), each TGFP isoform may have unique roles in embryo-uterine interactions during the process of implantation. Preparation of the uterus to the receptive state for implantation and decidualization of the stroma after initiation of implantation involve proliferation and differentiation of various uterine cell types, tissue remodelling, and extracellular

matrix formation (12,14,48,49). Thus, TGF@s are likely to play important roles in implantation processes. These growth factors may also be important in neutralizing immunorejection of the allogenic embryo by the maternal immune system (50). Furthermore, because of their role in mesoderm induction in Xenopus embryos (18, 19), uterine-derived TGF&s could be important in early mammalian embryogenesis (51-53). The expression of TGFPl and $2 in the uterine epithelium and deciduum is consistent with this suggestion. In contrast, the selective expression of TGF/33 in the uterine myometrium suggests more restricted functions for this TGF/? isoform. TGFPs have been suggested to be involved in angiogenesis, inhibition of vascular smooth muscle cell proliferation, and stimulation of cellular hypertrophy (54-56). However, expression of TGFP isoforms in smooth muscle cells has not been reported previously. Several lines of evidence suggest a role for TGFP3 in myogenesis. High levels of TGF/33 mRNA have been detected in embryonic and adult cardiac muscles (57-59), and recently, TGFP3 has been shown to be expressed at high levels in skeletal muscle and skeletal myoblast cell lines (59). TGFP3 mRNA levels increase after myoblast fusion. However, all three TGFP isotypes can inhibit myoblast fusion, and TGFPl can inhibit the expression of myogenin, a musclespecific gene (59-62). Interestingly, a loss of TGFp receptors occurs with muscle cell differentiation, which suggests that differentiated muscle cells may not be

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TGF-P,

I

I

I

I

I

I

I

I

wan*

LF

TGF-P,

Time after injection (h)

ILJJUI 06122461224

OVX

E2

E2+P,, D2

II-U 0 61224 ovx Pq

FIG. 4. Northern blot analysis of uterine TGFPP, TGFp3, and LF mRNAs in steroid-treated adult ovariectomized mice. Adult ovariectomized mice were injected with E, (250 ng), Pd (2 mg), or a combination of E, and P, at these doses, Uteri were collected 6, 12, or 24 h after injection. Poly(A)+ RNA (2.0 fig) was separated by formaldehydeagarose gel electrophoresis, transferred to nylon membranes, and hybridized to a 32P-labeled TGFP2 cRNA probe. After autoradiography for 48 h and quantitation of labeled probe bound by radioimaging, the membranes were washed to remove probe, and the blots were rehybridized with a 32P-labeled TGFP3 cRNA probe. After autoradiography for 24 h and quantitation of probe bound by radioimaging, the membranes were washed again and reprobed with a 32P-labeled LF cRNA probe followed by autoradiography for 6 h and quantitation of probe bound. A, Accumulation of TGFPZ, TGFP3, and LF mRNAs at the indicated times after an injection of E, or E, plus Pd. B, Accumulation of these mRNAs at the indicated times after an injection of P,. D2, Uterine poly(A+) RNA from D2 of pregnancy; OVX, poly(A)+ RNA from uteri of ovariectomized vehicle-treated (oil) mice. LF mRNA (2.6 kb) has been shown previously to be dramatically induced in the uterine epithelium in response to Ez, but not P, (41). Two major TGFp2 mRNA transcripts (6.0 and 4.0 kb) were transiently induced by E,.

responsive to TGFP (61). Preliminary experiments indicate the presence of three size classes of TGFp receptors in uterine myometrial membrane preparation (Das, S. K., and S. K. Dey, unpublished results). Although the expression of TGF/3s in a variety of target cells has been shown to be either up- or down-regulated by estrogen, progestin, testosterone, and antiestrogens (reviewed in Ref. 63), information regarding regulation of the expression of TGFP isoforms by steroid hormones in the uterus was not available. Ovarian steroid hormones are considered to be primary regulators of temporal and cell type-specific proliferation and differentiation in the uterus, processes hypothesized to involve growth factors (2-15). However, among the TGFP mRNAs detected in the uterus, the accumulation of only TGFp2 mRNA was transiently up-regulated by an injection of E2. Whether this transient accumulation of TGFP2 mRNA reflects an increased transcription rate or altered mRNA stability cannot be determined by the present study. The data establish that TGFP isoforms are differentially regulated in this organ. This finding is consistent with the concept that regulation of production

LF 14.0 7.0 6.0 3.0 1.0 6 12 24

6 1224

EZ

E2+P4

---

6 1224

tlme after lnjectlon

(h)

p4

FIG. 5. Quantitation of relative levels of uterine TGFP2, TGFP3, and LF mRNAs in steroid-treated ovariectomized mice. Northern blots used to produce the autoradiographs shown in Fig. 4 were analyzed using the radioanalytic image system (Ambis). Radioactive counts of probes hybridized to TGFP3 transcript (3.8 kb), LF transcript (2.6 kb), or two TGFP2 transcripts (6.0 and 4.0 kb together) were monitored for a period of l-2 h using the 32P-selective screen. The mean values from two independent experiments are presented as fold increase relative to the ovariectomized control value at different time points (6, 12, and 24 h) for the treatments with E,, Pq, or Ez and Pp. The mean net count for TGFPP in ovariectomized controls was 295/2 h, whereas that for TGFP3 were 253/h. Background counts over an equivalent area of the filter were subtracted from the experimental values. The ovariectomized controls are represented by dotted lines. OVX, Ovariectomy.

of TGFp isoforms by members of the steroid hormone superfamily is target tissue specific (63). The failure of an injection of E2 to influence accumulation of TGFPl (Das, S. K., G. K. Andrews, and S. K. Dey, unpublished results) or TGFP3 mRNA in the uterus cannot be explained solely on the basis of their cell type-specific expression, since uterine epithelial cells are responsive to this treatment in the adult mouse and express both TGFPl (12) and $2 genes. In summary, the differential pattern of expression of TGFp subtypes in the uterus suggests distinctive functions of these growth factors during early pregnancy. Acknowledgment Thanks are due to Crystal Gore for excellent technical assistance.

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