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Female Reproductive Tract |
Center for Animal Biotechnology and Genomics and Department of Animal Science, Texas A&M University, College Station, Texas 77843-2471
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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subunit, ßA subunit, ßB subunit, activin receptor (ActR) type IA, ActRIB, and ActRII in the developing ovine uterus. Ewes were ovariohysterectomized on PND 0, 7, 14, 21, 28, 35, 42, 49, or 56. The uterus was analyzed by in situ hybridization and immunohistochemistry. Neither inhibin subunit mRNA or protein was detected in the neonatal uterus. Expression of ßA and ßB subunits was detected predominantly in the endometrial LE and GE and myometrium between PND 0 and PND 56. In all uterine cell types, ActRIA, ActRIB, and ActRII were expressed, with the highest levels observed in the endometrial LE and GE and myometrium. Between PND 0 and PND 14, follistatin was detected in all uterine cell types. However, between PND 21 and PND 56, follistatin was only detected in the stroma and myometrium and not in the developing GE. Collectively, the present results indicate that components of the activin-follistatin system are expressed in the developing neonatal ovine uterus and are potential regulators of endometrial gland morphogenesis.
activin, developmental biology, follistatin, inhibin, uterus
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Activins and inhibins are members of the transforming growth factor (TGF) ß superfamily and regulate growth and differentiation of many branched epitheliomesenchymal organs via autocrine, paracrine, and perhaps, endocrine mechanisms [11–22]. Activins and inhibins are dimeric proteins [23, 24]. Activin consists of two ß subunits, ßA and ßB, that homodimerize or heterodimerize to form activin A (ßA:ßA), activin B (ßB:ßB), or activin AB (ßA:ßB). Inhibin consists of an subunit that heterodimerizes with an activin ß subunit to form either inhibin A (:ßA) or inhibin B (:ßB). The biological activity of activins is mediated by receptor complexes, consisting of activin receptor (ActR) type IA or ActRIB and ActRII [23–25]. One of the key features distinguishing the effects of activins from those of TGFß is that binding of activins to their receptors can be inhibited by follistatin and inhibin subunit [26–28]. Follistatin binds to activins with high affinity and neutralizes their activity [27, 29, 30]. Originally isolated from ovarian follicular fluid, follistatin inhibits pituitary FSH secretion [24, 31].
The activin-follistatin system is a complex regulatory system controlling cellular proliferation and differentiation in many epitheliomesenchymal organs, including the kidney, prostate, mammary gland, lung, pancreas, and salivary gland [11–22]. Our working hypothesis is that the activin-follistatin system exists in the uterus and, along with potential endocrine effects of hormones of the activin-follistatin system from the ovary, regulates endometrial gland branching morphogenesis in the neonatal ovine uterus. As a first step in testing this hypothesis, expression of the major components of the activin-follistatin system (follistatin, ßA subunit, ßB subunit, ActRIA, ActRIB, ActRII, and inhibin subunit) was studied in the uterus of the neonatal ewe.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The University Laboratory Animal Care and Use Committee of Texas A&M University approved all experimental and surgical procedures. Cross-bred Suffolk ewes were mated to Suffolk rams between September and November 2000. Pregnant ewes were maintained according to normal husbandry practices. Ewes included in the following experiments were born between January and May 2001. Ewes (n = 45) were assigned randomly at birth (PND 0) to be hysterectomized on PND 0 (n = 6), 7 (n = 4), 14 (n = 5), 21 (n = 5), 28 (n = 5), 35 (n = 5), 42 (n = 5), 49 (n = 5), or 56 (n = 5). The entire reproductive tract (uterus and ovary) was excised, and the uterus was trimmed free of the broad ligament, oviduct, and cervix. Sections from the middle of each uterine horn (thickness, 
1 cm) and half of each ovary were fixed in 4% (w/v) paraformaldehyde in PBS (pH 7.2). After 24 h, fixed tissues were changed to 70% (v/v) ethanol and then embedded in Paraplast Plus (Oxford Labware, St. Louis, MO). The remainder of the ovary and uterus was frozen in liquid nitrogen and stored at -80°C. The ovaries were analyzed in a companion study [32].
In Situ Hybridization
Partial cDNAs for follistatin, inhibin subunit, ßA subunit, ßB subunit, ActRIA, ActRIB, and ActRII mRNAs were generated by reverse transcription-polymerase chain reaction (RT-PCR) using total RNA isolated from the neonatal ovary or uterus as described previously [1]. Primer and annealing temperatures used for PCR are summarized in Table 1. The amplified PCR products were subcloned into the pCRII cloning vector using a T/A Cloning Kit (Invitrogen Life Technologies, Carlsbad, CA) and sequenced in both directions using an ABI PRISM Dye Terminator Cycle Sequencing Kit and ABI PRISM automated DNA sequencer (Perkin-Elmer Applied Biosystems, Foster City, CA) to confirm identity.
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Expression of mRNAs in uterine tissues was determined by in situ hybridization as described previously [33]. Briefly, deparaffinized, rehydrated, and deproteinated cross-sections (thickness, 5 µm) of the uterine horns from each ewe were hybridized with radiolabeled sense or antisense cRNA probes generated from linearized plasmid templates using in vitro transcription with [35S-]UTP. After hybridization, washing, and ribonuclease A digestion, slides were dipped in NTB-2 liquid photographic emulsion (Eastman Kodak, Rochester, NY), stored at 4°C for 2–28 days, and developed in Kodak D-19 developer. Slides were then counterstained with Gills modified hematoxylin (Stat Lab, Lewisville, TX), dehydrated through a graded series of alcohol to xylene, and protected with a coverslip. Images of representative fields of sections hybridized with antisense or sense cRNAs were recorded under bright- or dark-field illumination with a Nikon Eclipse 1000 photomicroscope (Nikon Instruments, Inc., Lewisville, TX) fitted with a Nikon DXM1200 digital camera using constant image acquisition parameters to ensure accurate comparison.
Immunohistochemistry
Expression of immunoreactive follistatin, inhibin subunit, ßA subunit, ßB subunit, ActRIA, ActRIB, and ActRIIA/B protein was detected in cross-sections (thickness, 5 µm) of the uterine horns from each ewe using specific antibodies and a Super ABC Mouse/Rat Immunoglobulin G (IgG) Kit (Biomeda, Foster City, CA) as described previously [34]. Mouse anti-human monoclonal antibody to follistatin (catalog no. MAB669), ActRIA (catalog no. MAB637), ActRIB (catalog no. MAB222), and ActRIIA/B (catalog no. MAB3391) were from R&D Systems, Inc. (Minneapolis, MN). Mouse anti-human antibody to inhibin subunit (catalog no. MCA951S), ßA subunit (catalog no. MCA950S), and ßB subunit (catalog no. MCA1661) were purchased from Serotec, Inc. (Raleigh, NC). The working antibody concentration employed for immunohistochemistry was 6.7 µg/ml for follistatin, 400 ng/ml for inhibin subunit, 200 ng/ml for ßA subunit, 200 ng/ml for ßB subunit, 1 µg/ml for ActRIA, 2 µg/ml for ActRIB, and 1 µg/ml for ActRII. Negative controls were performed in which the primary antibody was substituted with the same concentration of normal mouse IgG from Sigma Chemical Co. (St. Louis, MO). Antigen retrieval using boiling citrate buffer was performed for all antibodies according to the manufacturer's recommendations. The chromogen used for peroxidase localization was 3,3'-diaminobenzidine tetrahydrochloride from Sigma. Tissue sections from both uterine horns of each ewe were processed as sets within an experiment.
As described previously [3], relative staining intensity for immunoreactive protein expression was assessed visually in uterine sections (n = 2 per horn) from each ewe by two independent observers and scored as follows: absent (-; i.e., no staining above IgG control), weak (+), moderate (++), or strong (+++). The scores from the two observers were averaged. If histologically discernable, intercaruncular endometrial tissues (including LE, stroma, and GE) and caruncular endometrial tissues (including LE and stroma) and myometrium were scored. The GE was separated into shallow (stratum compactum) and deep (stratum spongiosum). Images of representative fields of sections probed with primary antibodies or IgG were recorded using a Nikon Eclipse 1000 photomicroscope fitted with a Nikon DXM1200 digital camera using constant image acquisition parameters to ensure accurate comparisons.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Partial cDNAs corresponding to inhibin subunit, ßA subunit, ßB subunit, ActRIA, ActRIB, and ActRII were generated by RT-PCR using total RNA from the neonatal ovine ovary or uterus and specific primers (Table 1). Analysis of sequences of the partial cDNAs verified their identity based on homology to sequences in GenBank of ovine or bovine origin (data not shown).
Inhibin Subunit, ßA Subunit, and ßB Subunit
Representative photomicrographs of in situ hybridization and immunohistochemistry results are presented in Figure 1. Patterns of immunoreactive protein expression are summarized in Table 2.
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Inhibin subunit
In the uterus, neither inhibin subunit mRNA or protein were detected by in situ hybridization and immunohistochemical analyses between PND 0 and PND 56 as compared to negative controls (data not shown). Although not detected in the uterus, abundant expression of inhibin subunit mRNA and protein was detected in granulosa and cumulus cells of antral follicles in the ovaries of the neonatal ewes in the present study (data not shown).
ßA subunit In the developing neonatal uterus, ßA subunit mRNA and protein were detected in all endometrial cell types but were most abundant in the endometrial LE and developing GE (Fig. 1A and Table 2). In the myometrium, ßA subunit expression was detected at low to moderate levels between PND 0 and PND 56. The uteri of some ewes contained black melanocytes that appear white in dark-field photomicrographs and black in bright-field photomicrographs of in situ hybridization and immunohistochemistry slides; however, these cells are not positive for mRNA or protein.
ßB subunit Expression of ßB subunit mRNA and protein was detected in all endometrial cell types, with the most abundant expression detected in the endometrial LE and GE (Fig. 1B and Table 2). In the myometrium, ßB subunit expression was detected at low levels.
ActRIA, ActRIB, and ActRII
Representative photomicrographs of in situ hybridization and immunohistochemistry results are presented in Figure 2. Patterns of immunoreactive protein expression are summarized in Table 3.
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ActRIA In the endometrium (Fig. 2A and Table 3), expression of ActRIA mRNA and protein was detected in all cell types but was most abundant in the endometrial LE and GE on PNDs 0, 7, and 56. In the myometrium, ActRIA was detected at low levels.
ActRII In the endometrium, ActRII mRNA and protein was detected in all cell types but was most abundant in the endometrial LE and ductal GE (Fig. 2B and Table 3). The antibody used for immunohistochemistry detects both ActRIIA and ActRIIB (R&D Systems). In the developing coiled and branched glands and myometrium, expression of ActRIIA/B protein was low.
ActRIB A partial cDNA for ActRIB was detected abundantly in total RNA isolated from the neonatal ovary but at extremely low amounts in the neonatal ovine uterus (data not shown). The low abundance of ActRIB mRNA was below the detectable limits of the in situ hybridization procedure (data not shown). In the endometrium, ActRIB protein was detected in all cell types but was most abundant in the endometrial LE and ductal GE (Fig. 2C and Table 3). In the developing coiled and branched uterine glands and myometrium, expression of ActRIB protein was low, except on PND 56.
Follistatin
Representative photomicrographs of in situ hybridization and immunohistochemistry results are presented in Figure 3. Patterns of immunoreactive protein expression are summarized in Table 4.
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In the neonatal uterus (Fig. 3 and Table 4), immunoreactive follistatin protein was abundant in the endometrial LE on PND 0 and PND 7. In contrast, only low levels of follistatin mRNA were detected in the endometrial stroma on PNDs 0 and 14. On PND 7 and PND 14, expression of follistatin protein was also detected in the emerging GE. However, by PND 21, follistatin expression was no longer detectable in the endometrial GE but had increased in the periglandular stroma and myometrium. Between PND 21 and PND 56, moderate to abundant levels of follistatin were detected in the endometrial stroma and myometrium but not in the developing GE.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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subunit, was detected in the endometrial LE and developing GE as well as in the uterine stroma and myometrium. Therefore, biologically active activins A, B, and/or AB likely are formed in the neonatal ovine uterus without interference from the inhibin subunit. Expression of both ActRI and ActRII was detected in the endometrial LE and GE as well as in the stroma and myometrium. Although inhibin subunit was not detected in the neonatal uterus, follistatin was expressed in the neonatal uterus, with highest levels detected in the endometrial stroma and myometrium between PND 21 and PND 56. Collectively, the results of the present study support our working hypothesis that the follistatin-activin system is present in the uterus and may have a regulatory role in postnatal uterine development during the critical period of coiling and branching morphogenesis of the endometrial glands. Components of the activin-follistatin system have been studied in various mutant mice [35]. However, widespread expression of activins in multiple organs and redundancy in the activin system have hampered efforts to determine the specific role of this system in the development and physiology of most organs. Female mice lacking the ßB subunit display abnormal mammary ductal elongation and alveolar morphogenesis during puberty and pregnancy [36]. Although mouse models indicate a vital role for activins in organ development, activins generally appear to inhibit epithelial morphogenesis [28, 37]. In these developing organs, activins disrupt normal lobulation patterns of epithelial growth in the developing pancreas and salivary gland [11]. In contrast, other studies indicate that activin may trigger digestion of the extracellular matrix at the site of a new branch and that activin A may cause breakdown of the extracellular matrix throughout the epithelium in the kidney [38]. However, activin A inhibits branching and elongation of epithelia in cultured mouse pancreatic rudiments, thereby blocking development of the exocrine pancreas [11]. Addition of exogenous activin A inhibits hepatocyte growth factor (HGF)-induced growth of mammary epithelial cells and tubule formation [16]. Interestingly, HGF and the HGF receptor, c-met, are candidate regulators of postnatal uterine development and adenogenesis in the neonatal ewe [3, 4]. The biological effects of activins are mediated by ActRI and ActRII. In the present study, ActRIA, ActRIB, and ActRII were detected in emerging, proliferating, and branching GE. Activins initially bind to a type II binding receptor, which then recruits and phosphorylates a type I signaling receptor. Two type I receptors are known, ActRIB and ActRIA [39–41]. In the present study, ActRII was detected in endometrial LE, GE, and stroma as well as myometrium. In contrast, ActRIA and ActRIB were expressed predominantly in endometrial LE and GE. Overall, the most abundant expression of the ActR subunits was in endometrial LE and GE. In the endometrial GE, expression was most abundant in the developing glands before PND 14 and on PND 56. Available results indicate that activins produced in neonatal ovine uterus may play distinct autocrine and paracrine roles in gland development during the periods of endometrial gland bud differentiation, elongation or tubulogenesis, and branching morphogenesis. The possibility of endocrine effects of these hormones from the ovary must also be considered [32]. Future experiments will explore the roles of activins and their receptors in neonatal ovine endometrial adenogenesis.
In other developing organs and in the endocrine system, the autocrine and paracrine activities of activins can be regulated by follistatin and the inhibin subunit [26, 27, 29, 30]. In human endometrium and developing rat prostate, expression of the inhibin subunit was not detected [15, 42]. Similarly, inhibin subunit mRNA and protein were either absent or below detectable limits in neonatal ovine uterine tissues in the present study. However, follistatin mRNA and protein were readily detected in the neonatal ovine uterus. Between birth and PND 14, follistatin protein was expressed in endometrial LE, GE, and stroma. Interestingly, follistatin mRNA was detected only at low levels in endometrial stroma between PNDs 0 and 14. After PND 14, follistatin expression declined to undetectable levels in endometrial LE and GE and increased in the endometrial stroma and myometrium. As for ActR subunits, these striking spatial changes in follistatin expression are clearly associated with the period of endometrial gland coiling and branching morphogenesis that occurs primarily between PND 14 and PND 56.
Follistatin has been implicated as a regulator of epithelial branching morphogenesis in a number of organs. The expression of follistatin is regulated by various factors, including activins [43]. In Madin-Darby canine kidney (MDCK) cells, epithelial tubulogenesis can be stimulated by HGF or addition of exogenous follistatin [36]. Indeed, stimulation of MDCK cell tubulogenesis by HGF, a stromal cell-derived growth factor expressed during ovine uterine adenogenesis [3], involves suppression of activin A expression [44]. In the developing prostate, follistatin can neutralize the inhibitory effects of exogenous activin A and appears to modulate the rate of branching morphogenesis [15]. A similar effect has been observed in the pancreas [18]. Available results indicate that a carefully orchestrated interplay between activin and follistatin is important for correct epithelial branching morphogenesis and that a similar system likely regulates endometrial gland branching morphogenesis in the neonatal ovine uterus. Indeed, a recent study by our laboratory found that ovariectomy of ewes on PND 7 retarded uterine growth as well as coiling and branching morphogenesis of endometrial glands on PND 56 [32]. In that study, the normal patterns of activin-follistatin system expression observed in the present study were significantly altered in the uterus of ovariectomized ewes. Given that the neonatal ovine ovary develops numerous antral follicles that express all the components necessary to produced activin, follistatin, and inhibin [32, 45, 46], these factors likely are secreted into the circulatory system and act in an endocrine fashion to influence uterine development by the activin-follistatin system [32]. Results from the present study support the working hypothesis that the activin-follistatin system is present in the developing neonatal ovine uterus and is involved in endometrial gland morphogenesis. Specifically, activins are expressed predominantly in the developing endometrial epithelium of the uterus. Between PND 0 and PND 14, activins may be involved in GE bud formation and tubulogenesis. After PND 14, follistatin expression decreases in the LE and developing GE and increases in the stroma and myometrium. During this period of coiling and branching morphogenesis, follistatin may bind epithelial activin subunits and prevent the inhibitory effects of activins on epithelial growth and differentiation. Future experiments will be directed toward understanding the role of the activin-follistatin system in branching morphogenesis of the endometrial glands during development of the neonatal ovine uterus.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Thomas E. Spencer, Center for Animal Biotechnology and Genomics, 442 Kleberg Center, 2471 TAMU, Texas A&M University, College Station, TX 77843-2471. FAX: 979 862 2662; tspencer{at}ansc.tamu.edu
Received: 12 February 2003.
First decision: 21 March 2003.
Accepted: 8 May 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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alters oestrogen receptor and oxytocin receptor expression in the endometrium of cyclic ewes. J Mol Endocrinol 1995 15:203-220 and ßA subunits in the ovaries of postnatal and prepubertal lambs. Reprod Fertil Dev 1997 9:825-832[CrossRef][Medline]This article has been cited by other articles:
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  P. Ciarmela, E. Wiater, and W. Vale Activin-A in Myometrium: Characterization of the Actions on Myometrial Cells Endocrinology, May 1, 2008; 149(5): 2506 - 2516. [Abstract] [Full Text] [PDF]
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 M. Manikkam, R. C. Thompson, C. Herkimer, K. B. Welch, J. Flak, F. J. Karsch, and V. Padmanabhan Developmental Programming: Impact of Prenatal Testosterone Excess on Pre- and Postnatal Gonadotropin Regulation in Sheep Biol Reprod, April 1, 2008; 78(4): 648 - 660. [Abstract] [Full Text] [PDF]
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 K. Hayashi, A. R O'Connell, J. L Juengel, K. P McNatty, G. H Davis, F. W Bazer, and T. E Spencer Postnatal uterine development in Inverdale ewe lambs Reproduction, March 1, 2008; 135(3): 357 - 365. [Abstract] [Full Text] [PDF]
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K. Hayashi and T. E. Spencer WNT Pathways in the Neonatal Ovine Uterus: Potential Specification of Endometrial Gland Morphogenesis by SFRP2 Biol Reprod, April 1, 2006; 74(4): 721 - 733. [Abstract] [Full Text] [PDF]
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K. Hayashi and T. E. Spencer Estrogen Disruption of Neonatal Ovine Uterine Development: Effects on Gene Expression Assessed by Suppression Subtraction Hybridization Biol Reprod, October 1, 2005; 73(4): 752 - 760. [Abstract] [Full Text] [PDF]
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K. Hayashi, K. D Carpenter, T. H Welsh Jr, R. C Burghardt, L. J Spicer, and T. E Spencer The IGF system in the neonatal ovine uterus Reproduction, March 1, 2005; 129(3): 337 - 347. [Abstract] [Full Text] [PDF]
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K. Hayashi, K. D. Carpenter, and T. E. Spencer Neonatal Estrogen Exposure Disrupts Uterine Development in the Postnatal Sheep Endocrinology, July 1, 2004; 145(7): 3247 - 3257. [Abstract] [Full Text] [PDF]
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K. D. Carpenter, K. Hayashi, and T. E. Spencer Ovarian Regulation of Endometrial Gland Morphogenesis and Activin-Follistatin System in the Neonatal Ovine Uterus Biol Reprod, September 1, 2003; 69(3): 851 - 860. [Abstract] [Full Text] [PDF]
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