Expression of Messenger Ribonucleic Acid for Inhibin Subunits and Ovarian Secretion of. Inhibin and Estradiol at Various Stages of the Sheep Estrous Cycle'.
BIOLOGY OF REPRODUCTION 49, 281-294 (1993)
Expression of Messenger Ribonucleic Acid for Inhibin Subunits and Ovarian Secretion of Inhibin and Estradiol at Various Stages of the Sheep Estrous Cycle' 23 H. ENGELHARDT, 3 K.B. SMITH, A.S. MCNEILLY, 4 and D.T. BAIRD ' 4 Department of Obstetrics and Gynaecology3 and MRC Reproductive Biology Unit Centrefor Reproductive Biology, University of Edinburgh, Edinburgh EH3 9EW, United Kingdom
ABSTRACT The relationship between expression of inhibin mRNA and ovarian secretion of estradiol (E2) and immunoactive inhibin was investigated at midluteal phase and throughout the follicular phase of the sheep estrous cycle. At laparotomy, timed samples of ovarian blood were collected and ovaries were removed from 39 Scottish Blackface ewes (ovulation rate 1.3 ± 0.1) on Day 10 of the luteal phase or 24, 48, 60, 72, or 84 h after injection of cloprostenol (PG; 100 $xg) on Days 10-12. Ovaries were removed 35 and fixed for in situ hybridization using S-labeled antisense riboprobes transcribed from inhibin a, pA, and PB cDNAs. LH, E2, and inhibin concentrations were determined by RIA. On the basis of peripheral LH levels and the presence of estrogen-active follicles (E-A; > 3 mm in diameter secreting > 1 ng/min E2) or recent ovulations, animals were grouped as follows: presurge (24 or 48 h post-PG; LH < 5 ng/ml; n = 7), midsurge (with E-A; LH > 5 ng/ml; n = 6), late surge (large follicle not E-A; LH > 5 ng/ml; n = 4), postsurge (large follicle not E-A; LH < 5 ng/ml; n = 7), and postovulation (n = 10). As expected, E2 secretion by the "active" ovary (containing preovulatory follicle) tended to increase with follicular development such that secretion was maximal at midsurge and then declined. E, secretion by the "inactive" ovary was low at all stages. Immunoactive inhibin, in contrast, was secreted in substantial quantities by both ovaries, although secretion from active ovaries was higher at all stages (p < 0.05). Effects of stage on secretion were not significant, but immunoactive inhibin secretion from active ovaries was high in postsurge animals when E, secretion was very low. Hybridization for inhibin mRNA was specific for granulosa cells of antral follicles. While most sheep in the luteal (4 of 5), presurge (2 of 3), and midsurge groups (5 of 5) had at least one inhibin-positive large follicle (expressing both a- and P-subunit mRNA), none were present between the LH surge and ovulation (late and postsurge groups). Inhibin mRNA was undetectable in midcycle CL, but 4 of 10 recent ovulations hybridized weakly with the a probe and one very weakly with the IA probe. The mean number of inhibin-positive large follicles per animal (in those having at least one) was 1.3 + 0.15 (n = 15 ewes). In most animals, a proportion of small follicles were either inhibinpositive or expressed a-subunit only, except in the midsurge and late/postsurge groups (inhibin-positive small follicles present in 1 of 5 midsurge ewes and in no late/postsurge ewes). No large follicles and very few small follicles (13 of 572 assessed) hybridized with one or both of the ,B probes but not the a probe. We conclude that 1) mRNA for inhibin a-, PA-, and P-subunits is expressed in large E-A follicles and in a minority of small antral follicles during both the follicular and luteal phases; 2) inhibition of expression of inhibin mRNA occurs at about the same time as the decline in E2 secretion after the LH surge; and 3) immunoactive inhibin secreted in the late follicular phase does not represent newly synthesized inhibin.
preovulatory follicle to grow and differentiate in spite of the reduction in FSH concentrations. Although inhibin secretion increases [7, 8] and FSH levels decrease [9] during the follicular phase in the sheep, this change in inhibin secretion does not seem to account for the fall in FSH [7]. Although the endocrine role of inhibin during this period is uncertain, evidence is accumulating to support the paracrine aspect of this hypothesis: in vitro studies in the rat [10] and human [11] have shown that inhibin enhances LHstimulated thecal androgen production. If a similar mechanism were to operate in vivo in follicles with already activated aromatase systems, estradiol secretion could conceivably increase in spite of decreased FSH levels. The aspect of the hypothesis that is least understood is the precise location where the inhibin is produced and the manner in which its production is regulated. On the basis of RIA of homogenates of ovarian tissue, granulosa cells have been determined to be the major site of inhibin synthesis in the sheep ovarian follicle [12]. To date, expression of inhibin mRNA in the sheep ovary has been investigated only through Northern blot analysis of RNA extracted from pools of follicles, corpora lutea, or stroma [13, 14]. These studies
INTRODUCTION Inhibin is a glycoprotein composed of two disulphidelinked polypeptide chains, termed a and 13, that have been shown to be the products of different genes [1, 2]. Two related 3-subunits have been identified [1, 3], which when associated with the a-subunit give rise to inhibin A (ta/,A) and inhibin AB (a/PB). It is well established that inhibin selectively inhibits FSH secretion by the anterior pituitary (for review see Lincoln et al. [4]), whereas dimers of the p-subunits (PA/PA, PA/PB; activin A or activin AB) can selectively stimulate FSH release and synthesis [5, 6]. In the sheep, a role for inhibin in the selection of the ovulatory follicle has been hypothesized. According to this hypothesis, inhibin secreted by the preovulatory follicle acts in an endocrine fashion to depress the secretion of FSH while concurrently having paracrine actions allowing the Accepted March 22, 1993. Received January 18, 1993. IThis work was supported by MRC Programme 892953. 2 Correspondence: Prof. D.T. Baird, Department of Obstetrics and Gynaecology, University of Edinburgh, 37 Chalmers Street, Edinburgh EH3 9EW, UK. FAX: (31) 229-2408.
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gave little or no information about cellular source or regulation of expression with respect to the cycle. In vivo experiments in the sheep have shown that the major source of inhibin is large estrogenic follicles [12,15], although unlike estradiol, inhibin is secreted in appreciable quantities by large, nonestrogenic follicles and small follicles [15]. Although inhibin secretion in the sheep can be stimulated by long-term treatment with FSH [7, 16], interpretation of these effects is confounded by the stimulatory action of FSH on follicular growth. In the short term, inhibin secretion has been shown to be unaffected for up to 12 h after injection of FSH in amounts that double basal concentrations [17]. Inhibin is secreted by the sheep ovary in pulses, but each pulse is unrelated to pulses of FSH or LH [18, 19]. In contrast to the situation in the sheep, there is in vitro evidence in other species demonstrating stimulation of inhibin production by FSH and testosterone (cow [20], human [21]). Inhibin production by human granulosa cells from large follicles is responsive to both FSH and LH while luteinized granulosa cells are stimulated by only LH [22], suggesting that both gonadotropins may regulate inhibin secretion depending on the stage of the cycle. A previous report in the sheep [23] demonstrated an increase in secretion of immunoactive inhibin after the LH surge at a time when estradiol secretion was markedly reduced, suggesting that the preovulatory LH surge had differential effects on synthesis of these two products. To investigate this possibility further, levels of immunoactive and bioactive inhibin were compared at various stages of the cycle. Immunoactive inhibin was quantified through use of an antibody directed against the a-subunit [7], and bioactivity was determined by inhibition of FSH production by ovine pituitary cells in vitro [24]. These two measures of inhibin were in general agreement at all times except after the LH surge, when apparent secretion of immunoactive inhibin was high but that of bioactive inhibin was low (unpublished results). One explanation of these findings was that the LH surge acted upon the preovulatory follicle to inhibit the expression of the 3subunit such that biologically inactive o-subunit, rather than dimeric inhibin, was being secreted at this time. Indeed, secretion of free -subunit has been previously described in cultured rat granulosa cells [25]. Little is known about the effect of the LH surge on smaller, nonovulatory follicles present on the ovary at the time of the LH surge. Clearly there are many gaps in our understanding of the regulation of inhibin synthesis in the sheep. The objectives of the current study were to 1) investigate changes in expression of mRNA for inhibin throughout the sheep estrous cycle using in situ hybridization and 2) relate these changes to ovarian secretion of estradiol and immunoactive inhibin throughout the same period. Our hypothesis was that the LH surge would inhibit the expression of mRNA for the 3-subunit of inhibin while maintaining or increasing expression of the -subunit.
MATERIALS AND METHODS Experimental Animals The experiments were conducted in accordance with the Animal (Scientific Procedures) Act of 1986 (UK) under Project License No. 60/00746. During the breeding season Uanuary 1992), estrous cycles of 39 mature Scottish Blackface ewes (ovulation rate 1.3 - 0.1) were synchronized by insertion of intravaginal progestagen sponges (Dunlop, Dumfries, UK) for 12-14 days followed by injection of 100 mg of the prostaglandin F,, analogue cloprostenol (PG; Estrumate, Dunlop) 12-14 days after sponge removal. Five animals underwent surgery 12-14 days post-PG (luteal phase). The remaining animals received a second PG injection 12 days after the first, and were allocated to five groups that underwent surgery 24 h (n = 6), 48 h (n = 11), 60 h (n = 6), 72 h (n = 6), and 84 h (n = 5) after the second PG injection. On the basis of previous experience with this breed of sheep, those ovariectomized at 24 h and 48 h post-PG were expected to be pre-LH surge. Animals in the last three groups received an injection of the GnRH agonist buserelin (10 mg; Hoechst, Hounslow, Middlesex, UK) 48 h after the second PG injection to ensure that an LH surge occurred [26]. Anesthesia was induced by minimal quantities (200-300 mg) of a mixture of two parts thiopentene (Intraval; RMB Animal Health Ltd., Dagenham, Essex, UK) and one part pentobarbitone sodium (Sagatal; May and Baker Ltd., Dagenham, Essex, UK) and maintained via halothane (1.5-2.5%). Midventral laparotomies were then performed, reproductive tracts were exteriorized, diameters of all large ( 3 mm) follicles were recorded, and both ovarian veins were cannulated using a 21-gauge needle. The proximal end of the ovarian vein was then occluded, and a timed 10-ml sample of ovarian blood was collected on both sides of the tract by gentle suction with a syringe. This procedure has been used extensively in our laboratory [15] and elsewhere [27]; ovarian secretion rates thus obtained are comparable with those from formal cannulations of the ovarian vein, both in situ [28] and after autotransplantation to the neck [29]. Jugular venous samples were taken by venipuncture between the two ovarian sample collections. Finally, both ovaries of each animal were removed and either fixed overnight in freshly prepared 4% paraformaldehyde in PBS, pH 7.4 (PFA), or used in related cell culture studies. Numbers of animals used for histology are indicated in Table 2. After fixation, ovaries were cut longitudinally into two or four slices; care was taken to cut through the largest follicles on each ovary. Tissue was then dehydrated and embedded in paraffin. Sections (5 ,um) were cut and mounted on slides coated with 3-aminopropyltriethoxy-silane (TESPA; Sigma, St. Louis, MO), incubated overnight at 60°C, and then processed for in situ hybridization as described below.
OVARIAN INHIBIN EXPRESSION IN SHEEP
Radioimmunoassays Plasma concentrations of LH, estradiol, progesterone, and inhibin were determined through use of previously established RLAs [7, 30-32]. The inhibin assay was based on an antibody raised against a synthetic fragment of the 1-26 amino acid sequence of the N-terminus of the ax-chain of 32-kDa porcine inhibin [33]. For this reason, biologically inactive free ot-subunit would cross-react completely in this assay. Although attempts to demonstrate free a-subunit in ovine follicular fluid [34] and ovarian venous plasma (K. Reddi, B.K. Campbell, and D.T. Baird, unpublished results) have been unsuccessful, the term "immunoactive inhibin" will be used to refer to the inhibin detected by RIA in order to allow for the possibility that all of what is being detected may not necessarily represent dimeric, biologically active inhibin. Concentrations of immunoactive inhibin were expressed as ng of the ot-peptide fragment. Values for inhibin levels obtained via the peptide assay in the present study would be approximately 1/12 of those obtained with the homologous bovine:bovine inhibin RIA in general use (standard = 32-kDa bINH-R-90/1 [35]). The sensitivities and intra- and interassay coefficients of variation (at 50% displacement of tracer) of the assays for LH, progesterone, estradiol, and inhibin were 0.2 ng (NIH-LH-S18)/ml--4%, 6%; 120 pg/ml-10%, 15%; 120 pg/ml-10%, 15%; and 35 pg/ ml-6%, 8%, respectively. Ovarian secretion rates of estradiol and inhibin were calculated by multiplying plasma concentrations by ovarian plasma flow rates (ovarian blood flow per minute corrected for hematocrit). Preparationof cRNA Probes From full-length human inhibin oa-, -, and B-subunit cDNAs [36], kindly provided by R.G. Forage, Sydney, Australia [2], selected sequence fragments of each inhibin subunit cDNA were chosen and subcloned into pBluescript SK(/ -) plasmids and subsequently transformed into competent XL I blue Escherichiacoli. (Saunders et al., unpublished data). The ot-subunit cDNA was 345 bp in length, coding part of the mature region of the human inhibin ao-subunit, corresponding to nucleotides 712-1057. The ,A-subunit cDNA was 447 bp in length, coding animo acids in the mature and pro regions of the P,-subunit protein precursors, complementary to nucleotides 670-1118. The P,-subunit cDNA was 558 bp in length, coding part of the inhibin P,B-subunit mature region and 3' untranslated region, corresponding to nucleotides 745-1303. 35 S-labeled sense and antisense riboprobes were generated from their cDNA templates by transcription using an RNA transcription kit (Stratagene, La Jolla, CA). The riboprobes were then degraded by partial alkaline hydrolysis to lengths of approximately 100 bp and were stored at -70°C. In Situ Hybridization The in situ hybridization procedure was performed as described by Wilkinson et al. [37,38]. Briefly, sections were
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deparaffinized in xylene and rehydrated through a series of alcohols, normal saline, and PBS. The sections were postfixed in PFA (20 min), washed, digested with proteinase K (20 mg/ml; Sigma, Poole, Dorset, UK) in 50 mM Tris, 5 mM EDTA buffer (TE), refixed in PFA, immersed briefly in sterile water and TE, and washed twice in 0.25% acetic anhydride in TE (10 min). The sections were then washed in PBS and saline, dehydrated through a series of alcohols, and air dried. Probes were diluted in hybridization mix (50% formamide, 10% dextran sulphate, 20 mM Tris-HC1, 5 mM EDTA, 10 mM sodium phosphate, 0.3 M NaCl, 0.02% Ficoll, 0.02% polyvinylpyrroline, 0.02% BSA, 0.5 mg/ml yeast RNA, 50 mM dithiothreitol [DTT]; all from Sigma) to obtain 1 x 105 dpm/ml; they were then heated at 80°C and cooled on ice before application to sections at 10 ,l/slide. After being overlaid with cover slips prepared from Gelbond film (FMC Bioproducts, Rockland, ME), slides were incubated overnight in a humidified chamber saturated with 5-strength SSC (Standard Saline Citrate: single-strength = 0.1 M NaCl, 15 mM sodium citrate) and 50% formamide at 55 0C. After hybridization, slides were washed in 5-strength SSC, 0.1 M DTT at 55°C for 30 min, then in double-strength SSC, 50% formamide, 0.1 M DTT at 65°C for 20 min. The slides were washed three times in NTE buffer (0.5 M NaCl, 10 mM Tris, 5 mM EDTA, pH 7.5) for 10 min at 37°C, treated with RNase (40 mg/ml in NTE; Stratagene) for 30 min at 37°C, and washed in NTE at the same temperature. The wash in double-strength SSC, 50% formamide, 0.1 M DTT (20 min, 65°C) was repeated, and the slides were then washed in buffers containing decreasing salt concentrations at room temperature (double-strength SSC, 0.1-strength SSC; 3 times, 10 min each). Slides were dehydrated through graded ethanols, air dried, and dipped in liquid emulsion (Kodak NTB-2 [Rochester, NY] diluted 1:1 in water immediately before use) at 40-42°C. Slides were exposed for 4 wk at 4°C, developed for 4 min in Kodak D-19 developer, washed for 1 min in water, and fixed for 4 min (Kodafix:water, 1:4). Slides were stained with hematoxylin (BDH, Glasgow, UK), mounted with Histomount (National Diagnostics, Highland Park, NJ), and subsequently examined and photographed under bright- and dark-field optics through use of a Zeiss microscope (Oberkochen, Germany). Sense riboprobes synthesized from each of the cDNAs were used on a selection of slides as a control for nonspecific binding. In all cases, background due to nonspecific binding was very low (not shown). StatisticalAnalysis Comparisons were made through either one-way or factorial analysis of variance after data were transformed logarithmically to reduce heterogeneity of variance. Statistical significance was inferred atp < 0.05. When the initial F test was significant, Fisher's Protected Least Significant Difference (PLSD) was used to compare individual means. Results are presented as untransformed means + SEM.
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FIG. 2. Ovarian secretion of estradiol and immunoactive inhibin (mean + SEM) from ovaries containing an estrogen-active follicle (E-A; 3 mm in diameter secreting > 1 ng/min estradiol; n = 18 ovaries), a large, nonE-A follicle ( 3 mm in diameter secreting < 1 ng/min estradiol; n = 35 ovaries), or small follicles only (< 3 mm; n = 25 ovaries). For each hormone, columns with different letters differ significantly (p < 0.05). (Data from different stages of the estrous cycle have been pooled.)
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