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Biology of Reproduction 62, 117-124 (2000)
©Copyright 2000 Society for the Study of Reproduction, Inc.

Article

Three Different Turkey Luteinizing Hormone Receptor (tLH-R) Isoforms II: Characterization of Differentially Regulated tLH-R Messenger Ribonucleic Acid Isoforms in the Ovary

Seungkwon Youa, Hyunggee Kima, Mohamed E. El Halawania, and Douglas N. Foster1,a

a Division of Animal Physiology, Department of Animal Science, University of Minnesota, St. Paul, Minnesota 55108


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We have recently characterized three different, alternatively spliced, partial turkey LH receptor (tLH-R) cDNA isoforms by the combination of reverse transcription-polymerase chain reaction (RT-PCR) and 5'- and 3'-rapid amplification of cDNA ends. The first cDNA (intact form: tLH-Rintact) showed 98% and 72–75% similarity with chicken and mammalian LH receptor sequences, respectively. The other two cloned cDNA isoforms (insertion and truncated forms: tLH-Rinsert and tLH-Rtrunc) could encode truncated soluble protein isoforms that lack the transmembrane region.

Northern blot analysis detected two transcripts of 3.0 kilobases (kb) (tLH-Rintact) and 1.5 kb (tLH-Rtrunc) in the turkey ovary but could not discriminate a third alternatively spliced transcript (tLH-Rinsert) due to the small 86-base pair difference in the size range of approximately 3.0-kb mRNAs. But with the combination of RNase protection assay, RT-PCR, and Northern blot analysis, three different alternatively spliced tLH-R mRNA isoforms were quantified. Differential expression of the tLH-R mRNA isoforms was demonstrated in ovarian stromal tissue during various reproductive stages and in the theca and granulosa layer through follicular development. To gain a better understanding of the physiological significance of the three different tLH-R isoforms, total RNA from the theca layer through follicular development after prolactin (PRL) treatment was analyzed by RT-PCR. PRL treatment for 8–14 days significantly increased the steady-state levels of total tLH-R mRNAs, including tLH-Rinsert and tLH-Rtrunc mRNAs, compared to those in nontreated controls. In contrast, the steady-state levels of tLH-Rintact mRNA during the same period was not significantly changed when compared to that in nontreated controls.

The present study shows that tLH-R transcripts are alternatively spliced in a tissue-specific manner in the turkey and that the mechanism may, in part, be controlled hormonally.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The pituitary secretion of the gonadotropic hormones FSH and LH is a highly regulated process that is important in folliculogenesis [1]. The ovarian follicle is completely dependent on the appropriate milieu of steroid and polypeptide hormones for its maintenance during the reproductive cycle. It is clear that the ability of the gonadotropins to modulate ovarian functions depends not only on the circulating levels of the gonadotropins but also on the expression of their receptors by potential target cells in the ovary [2].

Substantial progress has been made in elucidating the structures of the gonadotropin receptors by the cloning of mammalian LH receptor (LH-R) [3–5] and FSH-receptor (FSH-R) [6–8] cDNAs. Recently, chicken [9] and quail [10] partial LH-R cDNAs have been isolated. The chicken appears to have two different LH-R isoforms, one of which has an 86-bp insert located in the extracellular (EC) domain of the molecule [9]. Furthermore, three different, alternatively spliced, LH-R cDNA isoforms (intact, inserted, and truncated forms: LH-Rintact, LH-Rinsert, and LH-Rtrunc, respectively) have been characterized in the chicken and turkey [11]. Two of the cloned cDNA isoforms (LH-Rinsert and LH-Rtrunc) could potentially encode truncated protein isoforms lacking the transmembrane region that could be expressed as soluble proteins.

The regulation of LH-R has been shown to be dependent upon gonadotropins and prolactin (PRL). Several groups recently examined the hormonal regulation of LH-R mRNA in the ovary and demonstrated that the FSH-induced increase in LH-R is due, at least in part, to changes in the level of LH-R mRNA [12–14]. In certain physiological states when PRL is elevated, follicular development is retarded and estradiol production by the ovary is suppressed. For example, a marked increase in the circulating levels of PRL has been associated with incubation behavior, cessation of egg laying, and ovarian regression in the domestic turkey [15]. Exogenous administration of mammalian PRL has been shown to induce incubation behavior and/or gonadal regression and to cause a decline in circulating estrogen due to suppression in steroidogenic enzyme gene expression [15–19]. Ovarian regression may be achieved by a direct effect of PRL or may be attributable to reduced numbers of ovarian LH-Rs or both [18,19]. The biological actions of gonadotropins within the hen ovary are associated with gonadotropin-induced steroid synthesis and corresponding steroidogenic gene expression [20]. However, there is little information as to how gonadotropins and their corresponding receptors mediate this response.

Multiple LH-R isoforms also have been detected in mammalian species, suggesting a common mechanism(s) that may be evolutionarily conserved (at least between avian and mammalian species). Because the physiological significance of these transcripts is currently not understood, the primary objectives of this study were to evaluate their differentially regulated expression and to localize the turkey LH-R (tLH-R) transcripts in granulosa and theca tissue during follicular development. In addition, it is not known whether the different mRNA isoforms of tLH-R are under hormonal regulation or whether PRL suppresses tLH-R expression. This work is an initial step toward better understanding how PRL may impinge upon the differentially regulated expression of alternatively spliced tLH-R mRNA isoforms.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animal Studies

Nicholas Large White female turkeys were used throughout these studies. The reproductive groups (6–8 birds from each group) used were nonphotostimulated photosensitive (NPH), laying (LAY), incubating (INC), and photorefractory (REF) hens. Hens were routinely killed at the same time of day to avoid possible diurnal changes in hormone levels. Ovine PRL (NIADDK-oPRL-18; provided by the National Hormone and Pituitary Agency, Baltimore, MD) was injected into laying turkeys (4 mg [124 IU]/ml per bird) as detailed previously [21]. Theca layers from F1, F3, F5, and F7 follicles and the small white follicles (SWF) from each bird (for 0, 2, 4, 8, or 14 days) were collected and stored at -80°C for RNA analysis.

Northern Blot Analysis

To evaluate tissue-specific expression of the tLH-R transcripts, total cellular RNA was prepared from ovarian follicles, including ovarian stromal tissue (interstitial tissue plus follicles < 1 mm in diameter). In addition, morphologically normal and regressed follicles were collected and processed for RNA without separating granulosa and theca layers. Follicles were identified as regressed based on the presence of follicle haemorrhagia, collapsed morphology, and an opaque appearance [22]. Total cellular RNA was isolated from all tissues using Trizol reagent (Life Technologies, Gaithersburg, MD); 30–50 µg was separated by agarose gel electrophoresis in the presence of 6% formaldehyde, transferred to a nylon membrane (MSI, Westboro, MA), and then hybridized with a {alpha}-32P-labeled tLH-R cDNA insert at 42°C for 16–18 h. After hybridization, the membrane was washed twice (for 30 min each) with double-strength SSC, 0.1% SDS (single-strength SSC is 150 mM sodium chloride, 15 mM sodium citrate) at room temperature, followed by 2–3 washes (30 min each) in 0.1-strength SSC, 0.1% SDS at 65°C. Membranes were exposed to autoradiographic film at -70°C for 1–5 days. After autoradiography, the membranes were boiled for 30 min in 0.1-strength SSC/0.1% SDS to remove probe and rehybridized with chicken ß-actin insert [23], as previously described.

The band intensity of hybridization for tLH-R and ß-actin steady-state mRNA levels was quantified from autoradiographs using a scanning densitometer (Model 4000; Ambis, San Diego, CA). Results were normalized and compared with each other after dividing individual values by the intensity value of the ß-actin mRNA band plus the band intensity of the ethidium bromide-stained 28S and 18S rRNA. The rationale for this analysis is that ß-actin mRNA (a common housekeeping gene) was intended to be used as a quantity control for quantitative reverse transcription-polymerase chain reaction (RT-PCR) as described previously [24]. But ß-actin mRNA may not be expressed at consistent levels during hen follicle development; therefore a second standardization (28S and 18S RNA) was used to normalize values. Values were expressed as arbitrary densitometric units (ADU) or as percentage of control values.

RT-PCR

Single-stranded cDNA was synthesized from 2 µg total cellular RNA using oligo(dT) primer and reverse transcriptase, as recommended by the manufacturer (Perkin-Elmer/Cetus, Norwalk, CT). Reaction mixtures (50 µl) were prepared as described above; the amplification profile consisted of 24–34 cycles of 30 sec at 95°C, 30 sec at 65°C, and 30 sec at 72°C. The two different PCR products tLH-Rintact and tLH-Rinsert were tested to assure that the number of PCR cycles was in the linear range. These values were analyzed and normalized as described above (data not shown). The slope of the curve for the amplification of these two different products remained constant from 28 to 36 cycles, with quantification of products performed for every other cycle in 6 independent experiments. A similar amplification efficiency was observed for the total tLH-R or tLH-Rtrunc isoform when coamplified with ß-actin-specific fragments. The amplified products of the reaction were separated by electrophoresis on a 2% agarose gel, stained with ethidium bromide, and visualized (Eagle Eye II; Stratagene, La Jolla, CA), followed by transfer to a nylon membrane and Southern blot analysis as described above. The steady-state levels of tLH-R and ß-actin mRNAs by RT-PCR were quantified and normalized as described above.

Oligonucletide Primer Sequences

The ß-actin-specific oligonucleotide sequences [23,24] used in the PCR reaction were Act-1, 5'-TCT GGT GGT ACC ACA ATG TAC CCT; Act-2, 5'-ACC AGT AAT TGG TAC CGG CTC CTC. Different combinations of oligonucleotide primers were used for the amplification of total tLH-R transcripts (364 bp; S6F/S9R), coamplification of tLH-Rintact (416 bp) and tLH-Rinsert (502 bp) transcripts (splice-1/splice-2), and amplification of tLH-Rtrunc transcripts (325 bp) (trunc-1/trunc-2): S6F (106–130), 5'-CCA GAC TTG ACT CAG ATC TTC TTC T; S9R (446–470), 5'-GGC AGC CTC TTC AAT GAG TAC GAT G; splice-1 (446–470), 5'-GGC AGC CTC TTC AAT GAG TAC GAT G; splice-2 (838–862), 5'-AGT GGC TGG TTA TGA GGA CGA GGA G; trunc-1, 5'-CAT CGT ACT CAT TGA AGA GGC TGC C; trunc-2, 5'-CTG TGC AAC TTG TAA GGG TGA CTG A; trunc-4, 5'-TCA GTC ACC CTT ACA AGT TGC ACA G.

RNase Protection Assay (RPA)

RPA was performed according to the manufacturer's instructions (Ambion, Austin, TX). Briefly, a 420-bp fragment of the tLH-R from clone tLHR-420 was inserted into the pBluescript KS(+) vector (Stratagene) as previously described [25]. Antisense and sense RNA was transcribed in the presence of 50 µCi [{alpha}-32P]UTP using T7- and T3-RNA polymerase, respectively.

Approximately 105 cpm of the probe was coprecipitated with 50 µg of RNA samples from each of the tissues and then denatured at 65°C for 5 min; then complementary fragments were allowed to hybridize in the presence of hybridization buffer. The hybridization products were then digested with RNase A/T1 mixture (200 U/ml of RNase A and RNA T1) for 30 min at 37°C. The protected products were precipitated and analyzed by 5% denaturing acrylamide gel electrophoresis and then exposed to x-ray film as described above. The band intensity of the protected fragment was analyzed as delineated above.

Optimization of the RT-PCR assay utilized ß-actin as internal control [24], while Northern blots were normalized by the intensity of 28S/18S rRNA bands before and after transfer onto the nylon membrane; these normalized data were in turn compared to data used to normalize intensity of the ß-actin band. The three different methods used to normalize Northern blot data showed less than 5% difference in the values obtained (not shown). By Northern blot analysis, we could measure the relative portion of the tLH-Rtrunc isoform as a fraction of the total tLH-R transcripts. However, since Northern blot analysis could not separate tLH-Rintact from tLH-Rinsert mRNA, RPA was used to measure the relative proportion of the two different tLH-R isoforms. However, RPA analysis required 2–3 orders of magnitude more RNA than for the RT-PCR for quantification of the three different tLH-R transcript isoforms. On the other hand, RT-PCR had the versatility and resolving power to measure the steady-state levels of the three different isoforms of tLH-R mRNAs. When the three approaches were compared, these did not show any significant differences.

Statistical Analysis

All experiments were repeated at least two to five times. The data were examined by a one-way ANOVA using the General Linear Models procedure of the Statistical Analysis System (Cary, NC), and treatment means were compared by Duncan's Multiple Range test. Significance is reported at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Analysis of Steady-State Levels of Three Different LH-R mRNA Isoforms in Stromal Tissue from Various Reproductive Stages: Correlation of Data from Northern Blot Analysis, RPA, and RT-PCR

Northern blot analysis of tLH-R mRNA from stromal tissue detected two distinct 3.0-kilobase (kb) and 1.5-kb transcripts when the 5'-rapid amplification of cDNA ends (RACE) product that spanned the transmembrane (TM) and EC region was used as probe (Fig. 1A). However, the 1.5-kb transcript was not detected when the 592-bp PCR product (which contained only the TM domain) was used as a probe, suggesting that the tLH-Rtrunc isoform lacked the TM and intracellular (IC) regions (Fig. 1B). The band intensity of the ethidium bromide-stained 18S/28S rRNA bands (Fig. 1C) and the intensity of the ß-actin mRNA band (Fig. 1D) were used for normalization of equal RNA loading. Northern blot analysis detected two major transcripts from stromal tissue during different reproductive stages, with highest steady-state levels of total tLH-R mRNA in NPH (Fig. 1, A and B, lane 1) followed by REF (lane 4), LAY (lane 2), and INC (lane 3).



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  		FIG. 1. Representative Northern blot analysis of tLH-R mRNA from stromal tissue at various reproductive stages. Northern blots were prepared to measure the steady-state levels of stromal tLH-R mRNA from the reproductive groups NPH (lane 1), LAY (lane 2), INC (lane 3), and REF (lane 4). Blots were probed with either the 5'-RACE product (EC domain, A) or the 592-bp PCR product (TM domain, B). To normalize for equal RNA loading, the band intensity of ethidium bromide-stained 18S/28S ribosomal RNA (C) and the band intensity of ß-actin mRNA (D) were used

Although the Northern blot data could detect an approximate 3.0-kb tLH-Rintact transcript, it was not possible to discriminate between this isoform and the tLH-Rinsert isoform due to the small 86-bp difference. However, detection and quantification of both tLH-R isoforms were achieved by RPA and RT-PCR. Figure 2A shows a schematic diagram of the location of the antisense riboprobe that should protect three different fragments by RPA. Three distinctive bands (186, 256, and 490 bp for the tLH-Rtrunc, tLH-Rintact, and tLH-Rinsert isoforms, respectively) were protected using 50 µg of stromal RNA with antisense riboprobe (Fig. 2B). Yeast RNA was used as a control for hybridization to the riboprobe and did not protect the riboprobe in Figure 2B. Figure 2C summarizes data from RPA, RT-PCR, and Northern blot analysis. The decreasing order of steady-state levels of tLH-Rintact was NPH >= REF >= LAY > INC; that for tLH-Rinsert was NPH >= REF > INC >= LAY; and that for tLH-Rtrunc was NPH >= REF > LAY >= INC. However, the relative proportion of tLH-Rtrunc transcript as a percentage of the total tLH-R transcripts as shown in Figure 2D was 35 ± 14% (NPH), 37 ± 11% (LAY), 21 ± 9% (INC), and 49 ± 9% (REF). Similarly, relative proportions of tLH-Rintact and tLH-Rinsert mRNAs were represented as a percentage of total tLH-R mRNAs (Fig. 2D).



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  		FIG. 2. A) Schematic diagram of the antisense riboprobe and the expected sizes of three different protected fragments by RPA. B) A representative RPA using total RNA from different reproductive stages of stromal tissues. Three protected distinctive bands in sizes of 186, 256, and 490 bp are shown for tLH-Rtrunc, tLH-Rintact, and tLH-Rinsert transcripts, respectively. C) The band intensity of three protected fragments was analyzed, correlated with Northern blot and RT-PCR data, and represented as ADU. D) The relative proportion of each tLH-R isoform was analyzed and represented as percentage of total tLH-R. Data are expressed as the mean ± SEM; means with different letters significantly differ (P < 0.05)

Quantification of Alternatively Spliced tLH-R Isoforms During Follicular Development

To determine the regulated expression of alternatively spliced tLH-R isoforms during follicular development, the steady-state levels of tLH-R mRNA isoforms were analyzed by both RT-PCR and Northern blot analysis and were found to be in close agreement. The steady-state levels of total tLH-R mRNA tended to increase throughout follicular development of the theca layer (Fig. 3A; lane 3, F5 follicle; lane 2, F3 follicle; lane 1, F1 follicle). The steady-state levels of total tLH-R and tLH-Rinsert mRNAs in theca tissue between F3 and F1 were not significantly different. However, a significant decrease in the steady-state levels of LH-Rintact mRNA, and a significant increase in the levels of steady-state tLH-Rtrunc mRNA, was observed in theca layers from the F3 to F1 follicle (Fig. 3B). By comparison, there was an increase in granulosa cell tLH-Rintact mRNA levels during follicular development (Fig. 4, A and B) with a close correlation of total tLH-R mRNAs. Furthermore, Northern blot and RT-PCR analyses (Fig. 5, A and B) showed dramatic changes in total tLH-R mRNA levels between morphologically normal F3 follicles (lane 1) and regressed follicles of the same size (lane 2) (17.6 ± 2.5 ADU in normal versus 3.4 ± 5.2 ADU in regressed follicles). The steady-state levels of the three different isoforms of tLH-R transcripts were significantly lower in regressed follicles than in normal follicles (Fig. 5B).



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  		FIG. 3. A) Steady-state levels of tLH-R mRNA during follicular development of the theca layer by Northern blot analysis. Two distinctive 3.0-kb and 1.5-kb transcripts are shown in the representative autoradiograph with lanes as follows: F1, lane 1; F3, lane 2; and F5 follicle theca layer, lane 3. B) Normalized data from RT-PCR were correlated with data from Northern blot analysis and represented as fold change compared to values for F5 theca layer. Data are expressed as the mean ± SEM; means with different letters significantly differ (P < 0.05)



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  		FIG. 4. A) Top: Northern blot analysis of steady-state tLH-R mRNA levels during follicular development of granulosa layers. A representative autoradiograph, displaying two distinctive 3.0-kb and 1.5-kb transcripts, is shown as follows: F5, lane 1; F3, lane 2; and F1 follicle granulosa layer, lane 3. Bottom: Ethidium bromide-stained RNA gel. B) Normalized data from RT-PCR were correlated with data from Northern blot analysis and represented as fold change compared to values for F5 granulosa layer. Data are expressed as the mean ± SEM; means with different letters significantly differ (P < 0.05)



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  		FIG. 5. A) Representative autoradiograph of the steady-state levels of tLH-R mRNA from normal follicle (lane 1) and regressed follicle (lane 2). B) Band intensities from Northern blot analysis were normalized, correlated with data from RT-PCR, and represented as percentage of normal. Data are expressed as the mean ± SEM; means with * significantly differ from normal (P < 0.05)

Effects of Exogenous oPRL on the Expression of Three Different tLH-R mRNA Isoforms During Follicular Development

To determine whether the different tLH-R isoforms were regulated by PRL, laying turkeys were injected with oPRL. Total RNA was isolated from the theca layers of F1, F3, F5, F7, and SWF from each bird and analyzed by RT-PCR. The steady-state levels of tLH-Rinsert and tLH-Rtrunc isoforms in the F1 follicle increased significantly after the 4–14 days of oPRL injections (Fig. 6, A and D; lanes 1–5 in A represent Days 0, 2, 4, 8, 14, respectively). In contrast, there was no significant change in the steady-state levels of tLH-Rintact mRNA during the same period, even though some samples showed a clear decline in the abundance of tLH-Rintact mRNA with time of oPRL-treatment (Fig. 6, C and D). Similar results were observed in the steady-state levels of the three different tLH-R mRNA isoforms from the theca layers of F3, F5, F7 (data not shown), and SWF (Fig. 6E) after oPRL injection.



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  		FIG. 6. A–C) RT-PCR analysis of three different tLH-R mRNA isoforms using total RNA from F1 theca layer after oPRL treatment. Lane 1) 0-day oPRL treatment (control); lane 2) 2 days; lane 3) 4 days; lane 4) 8 days; and lane 5) 14 days of oPRL-treatment. D) The band intensities of amplified tLH-R mRNAs were analyzed, normalized, and represented as percentage of control. E) Summaries of steady-state levels of three different tLH-R mRNA isoforms by RT-PCR using total RNA from SWF after oPRL treatment. Data are expressed as the mean ± SEM; means with * significantly differ compared to 0-day oPRL treatment (P < 0.05)


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Three different tLH-R cDNA isoforms (tLH-Rintact, trunc, insert) were recently isolated from the turkey ovary and identified by nucleotide sequence analysis of RT-PCR and 5'- and 3'-RACE products. Partial characterization of the tLH-R genomic sequence could determine the origin of the tLH-Rtrunc and tLH-Rinsert isoform transcripts that potentially could produce truncated tLH-R protein isoforms. It is interesting to note that common mechanisms must exist among avian and mammalian species for producing soluble or secreted binding proteins from a single LH-R gene due to alternative mRNA splicing to produce proteins that contain only the ligand-binding domain (truncated isoform).

Multiple LH-R mRNA transcripts have been detected in mammalian species, and the size and relative abundance are both tissue- and species-specific [3,12,26,27]. In the rat ovary, the predominant mRNA is 6.7 kb in length with less abundant 1.2-, 2.6-, and 4.3-kb transcripts [27]. By comparison, both the ovary and testis of swine express LH-R mRNA in 1.4-, 2.6-, 4.0-, 4.7-, 5.8-, and 6.7-kb lengths but in different relative proportions [3]. While the physiological significance of these multiple transcripts is currently not understood, it has been demonstrated that the 1.2-kb LH-R mRNA from the rat encodes a truncated form of the receptor [27]. By contrast, there appears to be a single 3.0-kb LH-R transcript expressed in chicken ovarian tissues [9] and in quail testis [10]. Nevertheless, the identification of an alternatively spliced LH-R mRNA transcript containing an in-frame stop codon strongly suggests that the steady-state levels of cLH-R mRNA observed in the theca layer and granulosa cells during follicular development could be the summation of two alternatively spliced cLH-R transcripts (LH-Rintact and LH-Rinsert), since Northern blot analysis could not discriminate between the 86-bp difference in size [9,10].

Northern blot analysis of turkey ovarian tissue revealed that in addition to a predominant 3.0-kb tLH-R transcript, a distinctive 1.5-kb tLH-R transcript was also expressed when the 5'-RACE product (which contained the EC domain) was used as probe. But the same 1.5-kb tLH-R transcript was not detected when the 592-bp PCR product (which contained only the TM domain) was used as probe. This 1.5-kb tLH-R mRNA (tLH-Rtrunc) was found to encode a truncated form of the tLH-R that lacked both the TM and IC domains as confirmed by RPA, cloning, and sequence analysis (unpublished results). By comparison, the 1.2-kb rat LH-R mRNA also has been shown to encode a truncated form of the LH-R.

Because alternative splicing appears to be an important mechanism for the synthesis of variant LH-R proteins, the relative levels of the three different tLH-R isoforms were measured utilizing sensitive RT-PCR analysis. The steady-state levels of total tLH-R mRNA (as measured by either Northern blot, RPA, or RT-PCR) gradually increased within theca layers during follicular development, and this was generally consistent with all three tLH-R mRNA isoforms. On the other hand, there was a significant decrease in the steady-state levels of tLH-Rintact isoform mRNA in the F1 compared to the F3 follicle theca layers (in contrast to a slight increase in both tLH-Rinsert and tLH-Rtrunc transcripts). This decline in the tLH-Rintact transcript corresponded to a decreased steroidogenic responsiveness of F1 follicle theca layers to LH compared to that of F3 follicle. While this decline in steroidogenesis (decreased C17–20 lyase and aromatase activity) has previously been attributed to the loss of LH-stimulated steroidogenesis in mature theca cells [28], the present data suggest that there may be a concomitant decrease in tLH-Rintact expression. By comparison, in the chicken, the responsiveness of granulosa cells to LH stimulation, in terms of enhanced cAMP and steroidogenesis, showed a marked increase during the last few days of follicular maturation, peaking shortly after ovulation [29,30]. Also, the increased LH responsiveness of these cells during follicular maturation correlates well with significant increase in the tLHRintact expression. Taken together, the data suggest the possible physiological relevance of the three differentially expressed tLH-R isoforms (especially the tLH-Rinsert and tLH-Rintact isoforms) during follicular development in the hen ovary. The results that showed significantly decreased steady-state levels of not only total tLH-R mRNA but also of the three different isoforms of tLH-R transcripts in regressed follicles were not surprising, since similar results have been reported in the chicken ovary [9].

A marked increase in circulating PRL concentration is associated with incubation behavior, cessation of egg laying, and ovarian regression in the domestic turkey [15]. Exogenous administration of oPRL was shown to induce incubation behavior [31–33] and/or gonadal regression, reduce circulating estradiol, and suppress p450 aromatase gene expression [21].

PRL treatment for 8–14 days significantly increased the steady-state levels of total tLH-R mRNA (including both tLH-Rinsert and tLH-Rtrunc mRNAs). In contrast, the steady-state levels of the tLH-Rintact mRNA during the same period of time were not significantly changed when compared to those in nontreated controls. The effects of PRL on the steady-state levels of total tLH-R mRNA (including tLH-Rinsert and tLH-Rtrunc transcripts) were even greater in the small-size follicles (F3, F5, and F7, as well as SWF) than in the F1 follicles. It is important to compare these results with the study showing that PRL treatment significantly decreased circulating serum estradiol, testosterone, and LH levels without affecting serum progesterone levels [21]. Furthermore, the decreased levels of circulating steroid hormones were, at least in part, correlated with the reduced steady-state mRNA levels of cytochrome p450 aromatase in response to PRL (and this suppressive effect of PRL was much greater in F7 than in F1) [21]. In addition, high levels of PRL have been shown to inhibit ovarian follicular steroidogenesis by interfering with aromatase activity and by reducing androgen production from theca cells [16] and/or to act directly on granulosa cells to suppress progesterone and estradiol secretion through inhibition of steroidogenic enzymes [17]. This inhibitory effect of PRL on steroidogenesis might be attributable to reduced numbers of the ovarian intact form of LH-R in contrast to increased numbers of truncated LH-R isoforms. Furthermore, it was shown experimentally that induced hyperprolactinemic rats demonstrated impaired ovarian function that was associated with a significant reduction in available LH-R [18,19]. Two different alternatively spliced forms of tLH-R (tLH-Rinsert and tLH-Rtrunc) could be particularly important as receptor variants lacking the TM and IC region, since the secreted proteins would be capable of binding LH [34]. This soluble variant of the LH-R may be important in modulating extracellular LH levels by acting to reduce the concentration of free LH available to bind to the LH-R on target cells. The EC domains of these variants could act as competitive inhibitors of the native intact LH-R by serving as LH-binding proteins. For example, elevated LH levels have been implicated in several important biological processes including the preovulatory LH surge, the up- and down-regulation of LH-R expression, and receptor desensitization. Control of these functions may be related to the regulated synthesis of the soluble splicing variant through posttranscriptional and/or translational regulation of LH-R transcripts [35]. Thus, the antisteroidogenic effect of PRL at the ovary may be due to the down-regulation of the ovarian intact form of LH-R by increasing truncated LH-R isoforms, which leads to transcriptional changes in the steroidogenic enzymes 17{alpha}-hydroxylase and aromatase [21]. Furthermore, expression of the turkey PRL receptor in the ovary was recently characterized by Northern blot and RT-PCR (personal communication with Pitts et al.). In this context, it is possible that PRL could directly mediate the expression of alternatively spliced tLH-R transcripts at the level of the ovary during the follicular development of the theca layers.

Throughout follicular development, changes in the number of LH-R are tightly correlated with corresponding changes in the steady-state levels of LH-R mRNA that are mediated by a coordinated change in the transcription of multiple LH-R isoforms. This, in turn, suggests that the transcriptional and subsequent translational variants of LH-Rs are a major factor in regulating the levels of LH-R during follicular development [2]. In addition, this intriguing and complex link between follicular development and the regulated expression of LH-R isoforms could possibly be modulated by treatment with high levels of exogenous PRL.

Our present study showed tLH-R mRNAs are alternatively spliced in a tissue-specific manner that could be mediated by PRL. However, the molecular mechanisms underlying the hormonal regulation of alternatively spliced tLH-R transcripts in gonadal tissues are not yet understood, and clearly much more work needs to be done to resolve these important questions.


   FOOTNOTES
 
First decision: 14 September 1998.

1 Correspondence: Douglas N. Foster, Division of Animal Physiology, Department of Animal Science, University of Minnesota, 495 An Sci/Vet Med Building, 1988 Fitch Avenue, St. Paul, MN 55108. FAX: 612 625 2743; foste001{at}tc.umn.edu Back

Accepted: September 3, 1999.

Received: August 18, 1998.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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