BIOLOGY OF REPRODUCTION 58, (1998) JiaYin Liu, 3 Bruce. Aronow, 4, 7 David P. Witte, 5, 7 William F. Pope, 8 and Andrew R. La Barbera 2, 3, 6

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1 BIOLOGY OF REPRODUCTION 58, (1998) Cyclic and Maturation-Dependent Regulation of Follicle-Stimulating Hormone Receptor and Luteinizing Hormone Receptor Messenger Ribonucleic Acid Expression in the Porcine Ovary' JiaYin Liu, 3 Bruce. Aronow, 4, 7 David P. Witte, 5, 7 William F. Pope, 8 and Andrew R. La Barbera 2, 3, 6 Departments of Obstetrics and Gynecology, 3 Pediatrics, 4 Pathology and Laboratory Medicine,5 and Molecular and Cellular Physiology, 6 University of Cincinnati College of Medicine, Cincinnati, Ohio Children's Hospital Research Foundation,7 Cincinnati, Ohio Department of Animal Sciences, 8 Ohio State University, Columbus, Ohio ABSTRACT We sought to characterize the ovarian expression of mrnas for the receptors for FSH (FSHr) and LH (LHr) at the different stages of the estrous cycle of the pig and to localize the receptor mrnas to individual cell types in follicles and corpora lutea as a function of their developmental status. Northern blot analyses indicated that multiple FSHr and LHr mrna transcripts occur on Days 0, 4, 7, 12, and 16 of the estrous cycle. In situ hybridization analyses revealed considerable cyclic variation in expression among small, medium, and large follicles. In small follicles of both immature prepubertal ovaries and mature adult ovaries at all days of the cycle studied, FSHr mrna expression was strongly positive and was spatially restricted to granulosa cells. FSHr mrna expression was strongly positive in granulosa cells of medium follicles but had declined in granulosa cells of mature follicles on Day 0. LHr expression in small follicles, in contrast, was spatially restricted to theca cells and was estrous cycle-dependent. LHr expression in theca cells was weakly positive in small follicles on Days 12, 0, and 4, was strongly positive in small follicles on Day 7 and in medium follicles on Days 12 and 16, and had declined in large mature follicles on Day 0. LHr expression in granulosa cells was negative in small follicles on Days 4 and 7, weakly positive in medium follicles on Days 12 and 16, and positive in large follicles on Day 0. In degenerating follicles, LHr mrna was expressed weakly in the theca cells, and FSHr mrna expression in granulosa cells was absent. In the corpus luteum, FSHr mrna was not expressed, but LHr mrna was expressed in some cells on Days 4 and 7, was expressed maximally on Day 12, and had declined partially by Day 16. These studies indicate that there is maturation-dependent regulation of FSHr and LHr expression in the adult porcine ovary. FSHr mrna content decreases in parallel with FSHr protein content as follicles increase in size. INTRODUCTION FSH and LH are critical factors in folliculogenesis and ovulation in all mammalian species [1] including the pig [2, 3]. FSH and LH exert their actions on follicular cells via specific, high-affinity, membrane-bound receptors that are coupled to guanine nucleotide-binding proteins and cause activation of adenylyl cyclase [4]. Modulation of receptor number is one mechanism by which the responsiveness to gonadotropins is regulated in the developing follicle. Accepted October 20, Received April 4, 'Supported by grants from the National Institutes of Health (HD30370 to A.R.L. and DK47022 to B.J.A.) and the March of Dimes ( to B.I.A.). 2Correspondence: Andrew R. LaBarbera, University of Cincinnati Medical Center, P.O. Box , Cincinnati, OH FAX: (513) ; andrew.labarbera@uc.edu 648 Within the follicle, FSH receptors (FSHr) are localized exclusively in granulosa cells [5-7]. In the rat, the amount of FSH binding to granulosa cells appears to be a function of follicular size rather than of stage of the estrous cycle [8]. In the pig [9, 10] and the domestic hen [11], FSH binding is greater in small follicles than in larger follicles. In the cow, however, the converse is true, with large follicles exhibiting more FSH binding than small follicles [12]. In the pig, the decline in FSHr number with follicular growth is accompanied by a decline in the responsiveness of adenylyl cyclase to FSH [10, 13]. FSH binding in the corpus luteum has been demonstrated only in the hamster [5], cow [14], and human [7]. FSHr mrna has been localized only to granulosa cells of preantral and antral follicles and not to either theca cells or corpora lutea in both the rat [15] and cow [16]. Similarly, FSHr message was present in granulosa cells in small but not large follicles from either midluteal phase or preovulatory pig ovaries [17]. Abundant data indicate that the distribution of LH receptors (LHr) in the ovaries differs from that of FSHr. Autoradiographic studies revealed that in immature, preantral follicles of the rat, LHr are located primarily in theca cells, whereas in mature, preovulatory follicles, LHr also were present in granulosa cells [18]. In the ewe, usually only one follicle has LHr in both the theca and granulosa cells, and some follicles appear to have no LHr in either type of cell [19]. In the pig, LHr are detectable by radioreceptor assay in granulosa cells from small (< 3-mm) follicles, but are only about one twentieth the levels in preovulatory follicles [20, 21]. As porcine follicles enlarge, the adenylyl cyclase becomes increasingly responsive to LH or hcg [13]. LHr can be induced in granulosa cells by FSH both in vivo [18, 22, 23] and in vitro [24-26]. LHr mrna is expressed constitutively in theca cells of the rat and is induced by FSH in granulosa cells; after disappearing in response to LH at the time of ovulation, LHr reappears during formation of the corpus luteum [15, 27, 28]. On the basis of data obtained with follicles of different sizes during mid-diestrus and proestrus, it is apparent that LHr mrna expression increases in granulosa cells as follicles grow [17]. Pigs have numerous follicles that are synchronized at similar stages of development throughout the estrous cycle because they are multiple ovulators. The goals of the present study were, first, to characterize the expression and regulation of FSHr and LHr mrna in both follicles and corpora lutea throughout the estrous cycle of the pig, including early diestrus, mid-diestrus, late diestrus, early proestrus and estrus; second, to characterize the expression of FSHr and LHr mrna in prepubertal follicles; and, third, to de-

2 GONADOTROPIN RECEPTOR mrna IN PORCINE OVARY 649 termine whether FSHr mrna content increases or decreases with follicular maturation. MATERIALS AND METHODS Tissues Pairs of ovaries were removed surgically at laparotomy from either one prepubertal female gilt or one adult female gilt on each of Days 0, 4, 7, 12, and 16 of the estrous cycle. The onset of estrus was determined by observing for estrous behavior twice daily. Day 0 was considered to be the onset of estrus. One ovary of each pair was frozen immediately in liquid nitrogen and stored at C for extraction of RNA. The contralateral ovary was fixed overnight at 4 C in 4% (w:v) paraformaldehyde in PBS (0.14 M NaCl, 0.01 M sodium phosphate buffer, ph 7.4) for in situ hybridization. It then was transferred to 30% (w:v) sucrose in PBS and stored at 4 0 C for up to 1 wk. RNA Blotting The FSHr probe consisted of a 1005-basepair (bp) cdna encoding the transmembrane and intracellular portions of the porcine FSHr [29]; it had been isolated from p- Bluescript II KS + after digestion with EcoRI. The LHr probe consisted of a 783-bp cdna corresponding to nucleotides of the sequence published by Loosfelt et al. [30] with a 75-bp deletion from nucleotides ; it had been isolated from pbluescript II KS + after digestion with EcoRI and BamHI. The cdna inserts were random primer-labeled [31] using [- 2 P]deoxycytidine triphosphate (dctp) and the Kle- 3 now fragment of Escherichia coli DNA polymerase (Gibco, Grand Island, NY) and were purified by gel filtration chromatography on a Sephadex G50 column (Boehringer- Mannheim, Indianapolis, IN). Tissues stored at -80 C were weighed rapidly, pulverized, and then homogenized (0.1 g/ml) in TRI Reagent (Molecular Research Center, Montgomery, OH) using a Brinkman Polytron homogenizer (Brinkman Instruments, Westbury, NY). Proteins were extracted using phenol/chloroform, and total RNA was precipitated with isopropanol. Polyadenylated RNA was isolated using oligo(dt) cellulose columns (Sigma, St. Louis, MO). For qualitative Northern analysis [32], 5-7 Lg, based on spectrophotometric determination, of polyadenylated RNA were electrophoresed in 1% agarose gels containing 2.4 M formaldehyde. Electrophoresis buffer contained 5 mm EDTA, 10 mm sodium acetate, and 40 mm 4-morpholinepropanesulfonic acid (MOPS), ph 7.4. The section of the gel containing markers was cut and stained with ethidium bromide. RNA was transferred to Hybond TM-C nitrocellulose membrane (Amersham, Arlington Heights, IL) by blotting overnight with a 20-strength SSC (1.5 M NaCl, 150 mm trisodium citrate, ph 7.0) solution. RNA was bound to membranes by UV cross-linking. Membranes were prehybridized in 10 ml of hybridization solution containing 0.1% (w:v) BSA (Sigma), 0.1% (w:v) Ficoll (Pharmacia, Piscataway, NJ), 0.1% polyvinylpyrrolidone (Fisher Scientific, Pittsburgh, PA), 6-strength SSC, 1% (w:v) SDS (Fisher), 0.5 mg/ml sheared denatured calf thymus DNA (Sigma), and 0.1 M NaH 2 PO 4, ph 6.5, at 65 C for 2 h. Membranes were hybridized overnight at 65C with 1 x P-labeled cdna probe/ml hybridization solution. Membranes were washed with single-strength SSC containing 0.5% SDS at 65 C for 1 h and exposed to x-ray film (X-Omat AR; Eastman Kodak, Rochester, NY) for 4.5 days at -80 C with an intensifying screen. Membranes were stripped, and the hybridization procedure was repeated using a 404-bp human nonmuscle cytoskeletal -actin cdna [33] labeled with [a- 3 2 P]dCTP to determine the equivalence of RNA loading. In Situ Hybridization Sense and antisense complementary RNA probes were prepared by T3 or T7 transcription of linearized pbluescript KS + plasmids that contained FSHr or LHr cdna inserts described above using a transcription reaction kit (Stratagene, La Jolla, CA). Probes were synthesized using [ 3 5S]uridine triphosphate (UTP) (New England Nuclear, Boston, MA) at a specific activity of 1000 Ci/mM, treated with RNase-free DNase, extracted with phenol/chloroform, and purified by gel filtration using a Sephadex G50 Quick- Spin column (Boehringer-Mannheim). The radiolabeled probe was stored in 10 mm dithiothreitol (DTT) and 10 mm Tris HCl (ph 7.5) at -80 C for subsequent use in in situ hybridization. Ovarian tissue approximately 2 x 2 X 2 cm was fixed for 24 h at 4 C in 4% paraformaldehyde that was freshly prepared in single-strength PBS. The tissues were then equilibrated for 3-7 days in single-strength PBS that contained 30% sucrose, briefly equilibrated in O.C.T. embedding medium (Lipshaw, Pittsburgh, PA), and then frozen over a block of dry ice and stored at -80 C. The tissue was cut on a cryostat at 7- to 10-pxm thickness and placed onto 3-amino propyltriethoxysilane-coated slides (Histology Control Systems, Glen Head, NY), allowed to air-dry for 1 h, and then immersed in 4% paraformaldehyde for 1 h at room temperature. The slides were rinsed in 70% ethanol and briefly in double-strength SSC. Sections were incubated with 0.2 mg/ml proteinase K (Boehringer-Mannheim) in 0.2 M Tris-HCl (ph 8) for 5 min at 37 0 C; rinsed in Tris-glycine followed by double-strength SSC and acetic anhydride; dehydrated; prehybridized for 15 min at 42 0 C in 50% formamide, double-strength SSC, single-strength Denhardt's, 10% dextran sulfate, and 0.1 mm thio-deoxyuridine monophosphate (dump); and hybridized overnight at 42 C using 1 x 106 cpm of 3 5 S-labeled cdna probe in a 25-RI solution of the prehybridization buffer lacking thio-dump. The sections were treated with RNase, rinsed in doublestrength SSC containing 1 mm DTT and 50% formamide at 50 C with shaking, rinsed in double-strength SSC, and dehydrated in ethanol. Hybridized sections were coated with NTB2 emulsion (Eastman Kodak) and exposed for 14 days at 4 C, developed, and counterstained with hematoxylin and eosin (HE). Using darkfield optics, hematoxylinstained tissue appears red to purple and eosin-stained tissue appears dark to bright green. For each day of the estrous cycle, adjacent sections each were hybridized with either FSHr or LHr sense and antisense crna probes, respectively. Hybridization with sense probe served as negative control. Sections were examined using both brightfield and darkfield microscopy. RESULTS The estrous cycle of the pig normally is 21 days in length. The onset of estrus (the later preovulatory period) is considered Day 0 of the estrous cycle. Thus, Day 4 is early diestrus or the early luteal phase; Day 7 is mid-diestrus or the midluteal phase; Day 12 is late diestrus or the

3 650 LIU ET AL. FIG. 1. Northern blot analysis of FSHr and LHr mrnas in total ovarian tissue from one pig at each of the days of the estrous cycle indicated. Ovaries were removed from mature cycling sows on either Day 0, 4, 7, 12, or 16 of the estrous cycle. Five to 7 plg of polyadenylated RNA was loaded onto each lane. Blots were hybridized with either labeled FSHr cdna (A) or labeled LHr cdna (B) probe, washed, and exposed for 4.5 days. Sizes (in kb) of hybridized mrna transcripts relative to standard markers are indicated on the right of each panel. The membranes were stripped, rehybridized with labeled actin cdna probe (C, D), and exposed for 2 days. late luteal phase; and Day 16 is early proestrus or the beginning of the follicular phase. RNA Blotting RNA blot analysis of mrna from total ovarian tissue was performed to determine whether there were qualitative changes in the mrna transcripts for the FSHr and LHr during the estrous cycle of the pig (Fig. 1). On Days 0, 4, 7, 12, and 16 of the estrous cycle, the FSHr cdna probe hybridized to three bands on Northern blots (Fig. 1A). The bands corresponded to 4.2, 3.5, and 2.4 kilobases (kb). There were no differences in the sizes of the transcripts among the different stages of the estrous cycle. The 4.2-kb band predominated at all days. The variation in -actin cdna hybridization (Fig. 1, C and D) suggested that 3- actin mrna expression in the ovary varied during the estrous cycle. At Days 4, 7, 12, and 16 of the estrous cycle, the LHr cdna probe hybridized to seven bands on Northern blots (Fig. 1B) (Day 0 was excluded). The bands corresponded to 10.0, 6.8, 5.9, 4.7, 3.8, 2.5, and 1.5 kb. There were no differences in the sizes of the transcripts among the different stages of the estrous cycle. The 4.7-kb band predominated at all days studied. In Situ Hybridization The cellular localization of FSHr and LHr mrnas was studied by in situ hybridization. Adjacent sections of ovary obtained at each day of the estrous cycle were hybridized with antisense and sense crna probes. Hybridization with sense probes (negative control) for FSHr mrna (Fig. 2A) and for LHr mrna (Fig. 2B) was comparable to hybridization observed in nonfollicular or nonluteal regions of ovarian sections. Hybridization of both antisense and sense FSHr and LHr probes to spleen was comparable and thus served as a negative tissue control (not shown). Ovaries from prepubertal gilts have numerous small (< 3-mm-diameter) follicles. The mrna for the FSHr is expressed exclusively and strongly in the granulosa cells and cumulus cells of these follicles (Fig. 3A), whereas the mrna for the LHr is expressed exclusively in the theca interna (Fig. 3B). On Day 0 (estrus), ovaries contained both small and large (> 6-mm-diameter) follicles (Fig. 4). Large preovulatory follicles were observed only on Day 0. FSHr mrna signal was negative in the granulosa cell layer of large preovulatory follicles (Fig. 4A). LHr mrna signal was positive in both the theca interna and granulosa cells (Fig. 4B). Granulosa cells from less developed estrous follicles expressed substantial FSHr message (Fig. 4C), but no LHr message (Fig. 4D). Theca cells also expressed little or no LHr message (Fig. 4D). On Day 4, early diestrus, low levels of FSHr message were expressed in the granulosa cells of small antral follicles (Fig. 5A). LHr message was expressed at low levels in the theca interna but not in the granulosa cells of the same follicles (Fig. 5B). On Day 7, mid-diestrus, FSHr mrna hybridization was positive in granulosa cells of small antral follicles (Fig. 6A), and LHr mrna hybridization was positive in theca cells but negative in granulosa cells (Fig. 6B). Comparison of the follicle on the left with the follicle on the right in Figure 6C revealed that FSHr mrna was expressed in granulosa cells of some degenerating follicles (right) but not in granulosa cells of other degenerating follicles (left). Degeneration was established by examination of HEstained sections (not shown). LHr mrna expression was weak (Fig. 6D, left) to strong (Fig. 6D, right) in theca cells of the same follicles. On Day 12, late diestrnus, histologically normal mediumsized antral follicles (3-5-mm diameter) expressed FSHr mrna in the granulosa cells (Fig. 7A) and LHr mrna in

4 GONADOTROPIN RECEPTOR mrna IN PORCINE OVARY 651 PLATE I. FIG. 2. Nonspecific hybridization. As negative controls, tissue sections were hybridized in situ with sense crna probes for either FSHr (A) or LHr (B). Panels show darkfield views of a Day 4 small follicle (A) and a Day 7 corpus luteum (B). G, granulosa cell layer; T, theca cell layer; CL, corpus luteum. x90 (reproduced at 95%). FIG. 3. Localization of FSHr and LHr mrnas in small follicles of one immature pig ovary. Panels show darkfield views of adjacent tissue sections hybridized in situ with either antisense FSHr (A) or antisense LHr (B) crna probes. Nonspecific hybridization of sense crna probes was identical to hybridization in nonfollicular cells (not shown). G, granulosa cell layer; T, theca cell layer. x90 (reproduced at 95%). Bar = 0.1 mm and is the same for all sections. both the granulosa and theca cells (Fig. 7B). Figure 7, C and D, shows a degenerating follicle on the left and a healthy small follicle on the right. In the degenerating follicle, FSHr mrna expression in the granulosa cells was negative, and LHr mrna expression was negative in the granulosa cells but slightly positive in the theca cells. In the healthy follicle, FSHr and LHr mrna expression resembled that in Figure 7, A and B, respectively. On Day 16, early proestrus, FSHr mrna expression was strong in the granulosa cells of medium follicles and negative in other cell types (Fig. 8A). LHr mrna expression was strong in the theca cells but weak in the granulosa cells (Fig. 8B). Figure 9 illustrates the changes in gonadotropin receptor mrna expression in the corpus luteum during the porcine estrous cycle. For purposes of orientation, Figure 10 illustrates the identical HE-stained sections under brightfield optics. FSHr mrna expression was negative in the corpus luteum throughout the cycle as evident on Day 4 (Fig. 9A) and Day 12 (Fig. 9B). LHr mrna expression was positive but low in the corpus luteum on Day 4 (Fig. 9C) and Day 7 (Fig. 9D), reached a maximum on Day 12 (Fig. 9E), and had begun to decline by Day 16 (Fig. 9F). DISCUSSION Expression of mrnas for FSHr and LHr in the ovaries of prepubertal and mature pigs was studied by RNA blotting and in situ hybridization in order to determine the variation in mrna transcripts and the cellular distribution of receptor message expression. This is important because pigs are multiple ovulators with large numbers of follicles at different stages of development throughout the estrous cycle [34]. Qualitative RNA blot analysis revealed that three major FSHr mrna transcripts were present in the porcine ovary (Fig. 1A). The sizes of these transcripts agreed well with those reported previously for mrna extracted from cultured porcine granulosa cells and probed with porcine FSHr cdna, with the major transcript being 4.2 kb (Fig. 2A), which is in agreement with previous findings [29]. RNA blot analysis revealed seven major and minor transcripts of the LHr (Fig. B), two more minor bands than detected previously, with the major transcript being 4.7 kb (Fig. 2B), in agreement with previous findings [29]. The existence of multiple mrna transcripts for the gonadotropin receptors has been investigated by others. For LHr, variants of low

5 652 LIU ET AL. FIG. 4. Localization of FSHr and LHr mrnas in the ovary of one pig on Day 0 of the estrous cycle. Panels show darkfield views of adjacent tissue sections through either a large preovulatory follicle (A, B) or small follicles (C, D) hybridized in situ with either antisense FSHr (A, C) or LHr (B, D) crna probes. Nonspecific hybridization of sense crna probes was identical to hybridization in nonfollicular cells (not shown). G, granulosa cell layer; T, theca cell layer. x90. molecular weight can result from alternative splicing [35], and variants of high molecular weight can result from incorporation of 3' untranslated regions of the gene into mrna [361. It should be noted that the RNA used for Northern blot analysis was extracted from samples of total ovarian tissue and as such can be expected to represent a heterogeneous population of cells. This heterogeneity reinforces the importance of analyzing the distribution of the mrnas for FSHr and LHr with a technique such as in situ hybridization that localizes the mrnas to specific cell types in different ovarian structures. In situ hybridization analysis demonstrated that the distribution of FSHr and LHr mrnas changed dramatically during the estrous cycle and that follicles were very heterogeneous in their FSHr and LHr mrna content. In agreement with ligand autoradiographic studies [5-7] and in situ hybridization studies of rat [15], cow [16], and pig [17] ovaries, FSHr mrna localized only to granulosa cells in the developing follicle (Figs. 3-7). FSHr mrna was not evident in corpora lutea either on Day 4 (Fig. 9A), Day 7 (not shown), or Day 12 (Fig. 9B). This result agrees with the in situ hybridization results for the rat [15] and the cow [16] but appears to differ from the ligand autoradiographic results for the hamster [5], cow [14], and human 17]. The FSHr crna probe used in the present studies corresponded only to the transmembrane and intracellular domain regions of the receptor. Thus, our FSHr crna probe would have detected full-length FSHr mrnas but not alternately spliced partial FSHr mrnas encoding only the extracellular domain, which have been shown to be expressed in the bovine corpus luteum [37]. Expression of the full-length FSHr mrna is negligible in the mature bovine corpus luteum [37] in agreement with the present results. There have been conflicting reports of the changes in FSHr in granulosa cells during follicular growth and maturation. In rats [8] and sheep [19], binding of 25 1-FSH shows little change during the estrous cycle. In the pig [9, 10] and the domestic hen [11], FSHr decline as follicles enlarge. In the cow, some reports indicate that FSHr increase as follicles enlarge [12], some indicate that FSHr decrease [38, 39], and some indicate no change [40, 41]. The present observation that preovulatory follicles had little FSHr mrna (Fig. 4A) agrees with the previous reports that FSHr decrease as follicles enlarge, with the report that FSHr mrna levels in rat follicles decrease from metestrus to estrus [15], and with the previous report with pig follicles obtained in proestrus [17]. In the rat, FSHr mrna expression in granulosa cells of small follicles declined between proestrus and estrus [15], whereas in the present studies in the pig, FSHr mrna expression was strong in small follicles during both proestrus (Fig. 8A) and estrus (Fig. 4A). FSHr protein and FSHr mrna have been observed to in-

6 GONADOTROPIN RECEPTOR mrna IN PORCINE OVARY 653 PLATE II. FIG. 5. Localization of FSHr and LHr mrnas in the ovary of one pig on Day 4 of the estrous cycle. Panels show darkfield views of adjacent tissue sections through a small follicle hybridized in situ with either antisense FSHr (A) or LHr (B) crna probes. Nonspecific hybridization of sense crna probes was identical to hybridization in nonfollicular cells (not shown). G, granulosa cell layer; T, theca cell layer. x90. FIG. 6. Localization of FSHr and LHr mrnas in the ovary of one pig on Day 7 of the estrous cycle. Panels show darkfield views of adjacent tissue sections through medium (A, B) or small follicles (C, D) hybridized in situ with either antisense FSHr (A, C) or LHr (B, D) crna probes. Nonspecific hybridization of sense crna probes was identical to hybridization in nonfollicular cells (not shown). Degenerating follicles are shown in C and D. G, granulosa cell layer; T, theca cell layer. x90.

7 654 LIU ET AL. PLATE Ill. FIG. 7. Localization of FSHr and LHr mrnas in the ovary of one pig on Day 12 of the estrous cycle. Panels show darkfield views of adjacent tissue sections through medium follicles hybridized in situ with either antisense FSHr (A, C) or LHr (B, D) crna probes. Nonspecific hybridization of sense crna probes was identical to hybridization in nonfollicular cells (not shown). The follicle on the left in C and D was degenerating. G, granulosa cell layer; T, theca cell layer. x90. FIG. 8. Localization of FSHr and LHr mrnas in the ovary of one pig on Day 16 of the estrous cycle. Panels show darkfield views of adjacent tissue sections through a medium follicle hybridized in situ with either antisense FSHr (A) or LHr (B) crna probes. Nonspecific hybridization of sense crna probes was identical to hybridization in nonfollicular cells (not shown). GC, granulosa cell layer; T, theca cell layer. x90.

8 GONADOTROPIN RECEPTOR mrna IN PORCINE OVARY 655 FIG. 9. Localization of FSHr and LHr mrnas in corpora lutea of ovaries of single pigs during the estrous cycle. Panels show darkfield views of tissue sections through corpora lutea hybridized in situ with either antisense FSHr crna probe (A, B) on Days 4 (A) and 12 (B) or with antisense LHr probe on Days 4 (C), 7 (D), 12 (E), and 16 (F). Nonspecific hybridization of sense crna probes was identical to hybridization in nonluteal cells (not shown). CL, corpus luteum. x90. crease in vivo only in rats treated with either FSH [22, 42] or ecg [15, 43]. In vitro, FSH increases FSHr mrna levels in cultured rat granulosa cells [44]. In cultured porcine granulosa cells, FSH also increases FSHr mrna levels but simultaneously decreases membrane-bound FSHr [29]. In contrast, FSH causes a temporary suppression of FSHr mrna in cultured rat granulosa cells 2-6 h after exposure to hormone, followed by a recovery at 24 h [45]. Taken together, the results of previous studies and the results of the present studies suggest that while FSH may acutely increase FSHr mrna, follicular maturation is accompanied by a decrease in both FSHr mrna and protein. LHr mrna localized only to theca cells in small follicles from prepubertal ovaries (Fig. 3) and from estrous cycle Day 4 (Fig. 5), Day 7 (Fig. 6), and Day 16 (Fig. 8) ovaries. LHr mrna was expressed weakly in granulosa cells of some small follicles on Day 7 (Fig. 6D) and of medium follicles on Day 12. Small follicles on Day 16

9 656 LIU ET AL. FIG. 10. Brightfield views of the HEstained tissue sections through corpora lutea shown in Figure 9. The panels correspond exactly to those in Figure 9. A) Day 4; B) Day 12; C) Day 4; D) Day 7; E) Day 12; F) Day 16. CL, corpus luteum. 90. expressed little LHr mrna (Fig. 8B). At estrus, granulosa cells of preovulatory follicles expressed LHr mrna (Fig. 4B). These data are in agreement with previous studies which indicated that in immature follicles LHr are localized exclusively in theca [18]. The presence of LHr mrna in granulosa cells of large preovulatory (Fig. 4B) follicles but not of small follicles (Figs. 3D, 4B, 5B, and 7B) agrees with previous findings obtained with proestrous pig follicles [17] and with the findings of numerous investigators that FSH induces LHr in granulosa cells both in vivo in the rat [18, 22, 23] and in vitro in the pig [24, 26], the rat [25], and the mouse [46]. This finding also supports previous findings that treatment of granulosa cells in vitro with FSH or ecg increases LHr mrna in the rat [15, 28, 47] and the pig [29]. LHr mrna expression in the corpus luteum was low at both Day 4 and Day 7 (Figs. 8D and 9C) and reached a maximum on Day 12, the middle of the luteal phase of the estrous cycle. By Day 16, early proestrus, LHr mrna signal had decreased (Fig. 9F). In the rat corpus luteum, LHr expression is low soon after ovulation and luteinization, increases in the midluteal phase, and then declines in the late luteal phase [47]. The low levels of LHr mrna at the beginning of the luteal phase might reflect post-lh surge down-regulation followed by recovery. The decline in LHr mrna and protein levels late in the luteal phase probably are an early consequence of luteolysis [48] and contribute to the decline in LH responsiveness of the corpus luteum. In conclusion, these results, which are summarized in Figure 11, demonstrate that gonadotropin receptor expression in ovarian follicles of the pig during the estrous cycle is heterogeneous and depends both on follicular maturation and on stage of the estrous cycle. The relative abundance of these mrnas changes in total ovary during the estrous cycle, and in situ hybridization analyses indicate considerable cyclic variation in expression among mature small, medium, and large follicles. In small follicles of both immature prepubertal ovaries and mature adult ovaries at all days of the cycle studied, FSHr expression was strongly positive and was spatially restricted to granulosa cells. FSHr expression was strongly positive in granulosa cells of small and medium follicles (Fig. 11, I-VI) but had declined in granulosa cells of mature follicles at estrus (Fig. 11, VII). LHr expression, in contrast, was spatially restricted to theca cells and was estrous cycle-dependent. LHr expression in theca cells was weakly positive in small follicles on Days 12, 0, and 4 (Fig. 11, I-III), was strongly positive in small follicles on Day 7 (Fig. 11, IV) and in medium follicles on Days 12 and 16 (Fig. 11, V-VI), and had declined in large mature follicles on Day 0 (Fig. 11, VII). LHr expression in granulosa cells was negative in small follicles on Days 4 and 7, weakly positive in medium follicles on Days 12 and

10 GONADOTROPIN RECEPTOR mrna IN PORCINE OVARY 657 FIG. 11. Schematic diagram of cycleand maturation-dependent changes in FSHr and LHr in the porcine ovary. Follicles are represented by circles as follows: outer, theca cell layer; intermediate, granulosa cell layer; inner, the antrum. Solid circles represent CL; within each structure, left is FSHr and right is LHr. Stages of follicular maturation studied are numbered arbitrarily (I-XI). In a 21-day estrous cycle in which Day 0 is estrus, recruitment occurs in diestrus (luteal phase) between stages I and II. Throughout early to middiestrus (early to midluteal phase) follicles are small but growing (stages ll-iv). Numerous small follicles undergo atresia at this time. Medium follicles are seen in late diestrus (late luteal phase, stage V) and early proestrus (early follicular phase, stage VI). Large preovulatory follicles are observed at estrus, the onset of heat (stage VII). After ovulation, the residual granulosa cells luteinize and the follicles form corpora lutea (stages VIII-XI). 16, and positive in large follicles on Day 0. In degenerating follicles, LHr mrna was expressed weakly in the theca cells, and FSHr mrna expression in granulosa cells was absent. In the corpus luteum, FSHr mrna was never expressed, but LHr mrna was expressed in some cells on Days 4 and 7 (Fig. 11, VIII-IX), was expressed maximally on Day 12 (Fig. 11, X), and declined partially by Day 16 (Fig. 11, XI). REFERENCES 1. Greenwald GS, Roy SK. Follicular development and its control. In: Knobil E, Neill JD (eds.), The Physiology of Reproduction, Second Edition. New York: Raven Press; 1994: Foxcroft GR, Hunter MG. Basic physiology of follicular maturation in the pig. J Reprod Fertil Suppl 1985; 33: Esbenshade KL, Ziecik AJ, Britt JH. Regulation and action of gonadotropins in pigs. J Reprod Fertil Suppl 1990; 40: Richards JS, Hedin L. Molecular aspects of hormone action in ovarian follicular development, ovulation, and luteinization. Annu Rev Physiol 1988; 50: Oxberry BA, Greenwald GS. An autoradiographic study of the binding of 1 25 I-labeled follicle-stimulating hormone, human chorionic gonadotropin and prolactin to the hamster ovary throughout the estrous cycle. Biol Reprod 1982; 27: Charlton HM, Parry D, Halpin DMG, Webb R. Distribution of 125Ilabelled follicle-stimulating hormone and human chorionic gonadotrophin in the gonads of hypogonadal (hpg) mice. J Endocrinol 1982; 93: Shima K, Kitayama S, Nakano R. Gonadotropin binding sites in human ovarian follicles and corpora lutea during the menstrual cycle. Obstet Gynecol 1987; 69: Uilenbroek JT, Richards JS. Ovarian follicular development during the rat estrous cycle: gonadotropin receptors and follicular responsiveness. Biol Reprod 1979; 20: Nakano R, Sasaki K, Shima K, Kitayama S. Follicle-stimulating hormone and luteinizing hormone receptors on porcine granulosa cells during follicular maturation: an autoradiographic study. Exp Clin Endocrinol 1983; 81: LaBarbera AR. Follicle-stimulating hormone (FSH) receptors and FSH-responsive adenosine 3',5'-cyclic monophosphate production in porcine granulosa cells decline with follicular growth. Endocr Res 1994; 20: Ritzhaupt LK, Bahr JM. A decrease in FSH receptors of granulosa cells during follicular maturation in the domestic hen. J Endocrinol 1987; 115: Darga NC, Reichert LE Jr. Some properties of the interaction of follicle-stimulating hormone with bovine granulosa cells and its inhibition by follicular fluid. Biol Reprod 1978; 19: Lee CY. Adenylate cyclase of porcine granulosa cells: differential response to gonadotropins during follicle maturation. Endocrinology 1978; 103: Manns JG, Niswender GD, Braden T. FSH receptors in the bovine corpus luteum. Theriogenology 1984; 22: Camp TA, Rahal JO, Mayo KE. Cellular localization and hormonal regulation of follicle-stimulating hormone and luteinizing hormone receptor messenger RNAs in the rat ovary. Mol Endocrinol 1991; 5: Xu Z, Garverick HA, Smith GW, Smith ME Hamilton SA, Youngquist

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