Positive Association between Expression of Follicle-Stimulating Hormone ß and Activin ß_B-Subunit Genes in Boars¹

^a Endocrine Research Laboratory, Minneapolis Medical Research Foundation and Departments of Medicine, Hennepin County Medical Center and University of Minnesota, Minneapolis, Minnesota 55404 ^b UMDNJ-Robert Wood Johnson Medical School, Department of Neuroscience&Cell Biology, Piscataway, New Jersey 08854 ^c USDA, ARS, RLH U.S. Meat Animal Research Center, Clay Center, Nebraska 68933

	ABSTRACT

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This study tested our hypothesis that inhibin/activin (I/A) ß_B subunit and not follistatin (FS) gene expression relates positively to plasma FSH concentrations in the anterior pituitary gland of boars. Mature crossbred boars (n = 12) were selected for divergence in plasma FSH concentrations, and their anterior pituitary glands were evaluated for expression of the FSHß, I/A ß_B, FS, calmodulin, and GnRH receptor (GnRH-R) genes by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) and/or RNase protection assays (RPAs). Expression of I/A ß_B was greater (p < 0.01) in the six boars with high FSH than in the six with low FSH; expression of the I/A ß_B-subunit gene was positively correlated to that of the FSHß gene (RT-PCR: r = 0.96; p < 0.01; RPA: r = 0.68; p < 0.05). In contrast, expression of the FS (p > 0.10), GnRH-R (p > 0.08), and calmodulin (p > 0.10) genes was similar in the two groups of boars. Additionally, expression of the FSHß gene was correlated positively with pituitary and plasma FSH concentrations (r = 0.69 and 0.88, respectively; p < 0.05). These results support the hypothesis that activin B is partially responsible for elevated FSH concentrations in boars. Furthermore, the expression difference of the calmodulin gene observed previously between Meishan and White Composite boars represents a breed difference unrelated to FSH.

	INTRODUCTION

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One of the major objectives of importing Chinese pig breeds into the United States and European countries was to develop an understanding of the mechanism(s) of their increased prolificacy [1, 2]. FSH and LH are heterodimeric glycoprotein hormones composed of a common subunit and unique ß subunits that confer biological specificity to each hormone [3]. Both hormones are synthesized in the same cell type in the anterior pituitary gland [4]. The synthesis and release of these two pituitary glycoproteins are controlled by the complex interaction of multiple factors, including hypothalamic GnRH, sex steroids, opiates, and other peptides. GnRH is transported via the hypophysial portal circulation to the pituitary gland where it binds to specific high-affinity receptors (GnRH receptor; GnRH-R) and regulates LH and FSH secretion [5].

A long-standing problem in reproductive physiology concerns the differential or nonparallel secretion of FSH and LH [6] ostensibly from a single population of cells that respond to a single hypothalamic secretagogue. After elimination of the pulsatile GnRH signal from the hypothalamus by chemical, physical, genetic, or immunological means, pituitary gonadotrophs continue to secrete substantial amounts of FSH but very little LH in vivo [7, 8] or in vitro [9]. The phenomenon of GnRH-independent secretion of FSH was difficult to explain until the FSH-regulating proteins—inhibins, activins, and follistatin (FS)—were identified [10]. Inhibins and activins are structurally related dimeric glycoproteins, initially isolated from follicular fluids based on their ability to inhibit and stimulate, respectively, the release of FSH [11, 12]. Both inhibins and activins consist of two of three subunits, and ß_A or ß_B, while FS is a single peptide [13, 14].

Markedly greater plasma FSH concentrations and pituitary FSHß mRNA were detected in Meishan (MS) boars than in boars of contemporary U.S. breeds, but no such difference was observed between MS and White Composite (WC) females [15–18]. Previously we found greater expression of inhibin/activin (I/A) ß_B in anterior pituitaries of MS than in WC boars, but no differences were observed between these two breeds for the other genes screened [18]. It was unknown whether such a disparity was related to the elevated plasma FSH concentrations in MS boars or whether it was an intrinsic breed difference. In order to address this question, we selected two groups of MS x WC crossbred boars that had either high or low plasma FSH concentrations, and we evaluated expression of FSHß, I/A ß_B, FS, GnRH-R, and calmodulin genes. Calmodulin was included for evaluation because it was identified by differential display of pituitary cDNA as being different in pituitaries of MS and WC boars (unpublished results). Calmodulin is a regulator of intracellular calcium influx, and the response of pituitary gonadotrophs to GnRH is dependent upon calcium mobilization [19].

	MATERIALS AND METHODS

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Animal and Tissue Collections

Twelve mature 1/2 MS x 1/2 WC crossbred boars were selected from the U.S. Meat Animal Research Center swine population based upon their divergent plasma FSH concentrations, determined as the mean of three blood samples per boar, obtained by venipuncture when they were 4, 5, and 6 mo of age. Relative differences in FSH are maximal at this period of pubertal development [20]. These boars (6 with low and 6 with high plasma FSH concentrations) were progeny from 1/4 MS x 3/4 WC boars mated to 3/4 MS x 1/4 WC females or the reciprocal combination. Anterior pituitaries were obtained at slaughter at 14 mo of age, dissected into two halves, and frozen separately in liquid nitrogen immediately after removal from the animal.

FSH RIA

FSH concentrations were determined by RIA [20] in plasma samples obtained by jugular venipuncture at 4–6 mo of age and at 14 mo of age, the day prior to the day animals were killed, and in extracts of half of each anterior pituitary gland [17]. Antisera against ovine FSH (AFP-C5288113), ovine FSH for iodination (NIADDK-oFSH-17, AFP-6446C), and porcine FSH reference preparation (USDA B-1) were provided by the USDA/NIH National Hormone and Pituitary Program (Rockville, MD). The interassay coefficient of variation for pools of sera from intact or castrated males were 14.0% and 13.0% with mean concentrations of 296 and 1293 ng/ml, respectively.

RNA Isolation and Preparation

Total RNA was isolated from the remaining half of each pituitary gland by guanidine isothiocyanate extraction and CsCl centrifugation as described by Chirgwin et al. [21]. Prior to use, RNA was treated with RNase-free DNase I at 37°C for 30 min. After extraction with phenol-chloroform and ethanol precipitation, the RNA was redissolved into diethyl pyrocarbonate-treated water.

Primers Used for Semiquantitative Reverse Transcription-Polymerase Chain Reaction (RT-PCR)

Primers used for porcine FSHß, I/A ß_B subunit, FS, and ß-actin cDNA amplifications were the same as described previously [18, 22]. Based on the partial calmodulin cDNA sequence (290 base pairs [bp]) cloned from WC pituitary RNA by differential-display PCR in our laboratory [23], two primers with sequences of 5'-TGACAAGGATGGCAATGG-3' for the sense and 5'-CGGGAAAAAGGAGTTGAAAG-3' for the antisense were designed. The expected PCR product size amplified by this set of primers is 199 bp. Sense and antisense primers for the GnRH-R were 5'-GCTGCCTCTTCATTATCC-3' and 5'-AGACTGTGGGACAAATGG-3', which correspond to regions 668–675 and 906–923 nucleotides (nt) of the pig GnRH-R sequence, respectively [24]. The amplified PCR product size was expected to be 255 bp. All oligonucleotides used in this study were synthesized with an Oligo 1000 DNA Synthesizer (Beckman Instruments, Palo Alto, CA).

Subcloning and Sequencing

After synthesis of cDNA by reverse transcription of MS or WC boar pituitary RNA, PCR products were amplified with primers specific for the calmodulin and GnRH-R genes and were subcloned into the pCR-II vector (Invitrogen, San Diego, CA). The orientation of plasmids containing partial cDNA sequence was verified by sequence analysis from both directions by the dideoxy method of Sanger et al. [25], adapted for double-stranded DNA templates and T7 DNA polymerase (United States Biochemical Corp., Cleveland, OH). The sequence was compared with the corresponding sequence regions of each gene [23, 24].

Preparation of Oligonucleotide DNA, Sense and Antisense RNA Riboprobes

Oligonucleotide DNA was end-labeled with T4 polynucleotide kinase (Promega Corp., Madison, WI) in the presence of [-³²P]dATP and single-strength T4 polynucleotide kinase buffer (0.5 M Tris-HCl, pH 7.5, 0.1 MgCl₂). Free nucleotides were removed by Sephadex G-50 spin columns (5 Prime-3 Prime, Boulder, CO). Sense and antisense porcine FSHß and I/A ß_B-subunit riboprobes were transcribed in the presence of [-³²P]UTP with DNA-dependent T7 or SP6 RNA polymerases from the linearized plasmids with EcoRV or HindIII [18, 22]. The DNA templates were removed by incubation with RNase-free DNase I at 37°C for 15 min and extracted with phenol-chloroform.

Semiquantitative RT-PCR

Semiquantitative RT-PCR with 25 amplification cycles for the FSHß, I/A ß_B subunit, and ß-actin and with 30 cycles for FS cDNAs were conducted as described previously [18, 22]. The strategies used for determination of amplification cycles for the GnRH-R and calmodulin cDNAs were essentially the same [18, 22]. Briefly, 4 µl of cDNA mixture was amplified for 15 to 35 cycles under the identical conditions given in the previous papers. Based upon the exponential accumulation of radioactive PCR products, 30 amplification cycles were optimized with 0.2 µg total RNA for these two sets of primers. For each gene studied, the mRNA level of each sample was normalized to that of ß-actin mRNA, which was determined simultaneously under identical conditions except that different primers were used.

RNase Protection Assay (RPA)

Hybridizations and RNase digestions were performed with an RPA-II kit from Ambion Inc. (Austin, TX) under conditions described previously [18, 26]. Relative intensities of protected fragments were determined by transmittance scanning densitometry and were normalized with the protected fragment for an antisense, porcine ß-actin riboprobe.

Statistical Analysis

The mRNA level of each gene of interest was adjusted first to a constant amount of ß-actin mRNA. Student's t-test was used to determine differences between the high and low FSH groups. The correlative relationship between any two variables was determined by correlation analysis [27]. The data are presented as the mean ± SEM.

	RESULTS

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FSH Concentrations

Significant differences existed between these two groups of boars (p < 0.01; Fig. 1) in mean FSH concentrations in plasma samples obtained at 4–6 mo of age or on the day before slaughter at 14 mo of age, as well as in their pituitary extracts. Pituitary FSH concentrations were correlated to the mean of the three determinations of plasma FSH concentration obtained at 4–6 mo (r = 0.94, p < 0.01) and to the one obtained on the day before slaughter (r = 0.74, p < 0.05). Mean plasma FSH at 4–6 mo was also correlated with plasma FSH on the day before slaughter (r = 0.79; p < 0.05).

View larger version (38K): [in this window] [in a new window]   FIG. 1. FSH concentrations in boars that were selected for high or low FSH concentrations (n = 6 per group). Plasma was obtained at 4–6 mo of age and on the day before slaughter at 14 mo of age. Means within each FSH category that do not share a common letter differ (p < 0.01).

FSHß and I/A ß_B mRNAs (by RT-PCR)

The high and low FSH groups differed (p < 0.01) for FSHß and for I/A ß_B-subunit gene expression (Fig. 2) but not for the calmodulin gene (p > 0.10). Significant correlations were found between pituitary FSH concentrations and FSHß mRNA (r = 0.84; p < 0.05), between pituitary FSH concentrations and I/A ß_B-subunit mRNA (r = 0.74; p < 0.05), and between the FSHß and I/A ß_B-subunit mRNAs (r = 0.96; p < 0.01).

View larger version (28K): [in this window] [in a new window]   FIG. 2. Steady-state concentrations of mRNA, determined by RT-PCR, for the FSHß, I/A ßB-subunit, and calmodulin genes in anterior pituitary glands of boars that differed in FSH concentration. Means within each gene that do not share a common letter differ (p < 0.01).

FSHß and I/A ß_B Expression Levels (by RPA)

A schematic diagram of the RPA for the FSHß riboprobe with its expected protected fragment is given in Figure 3A. When the FSHß riboprobe was hybridized with 10 µg of pituitary total RNA and subsequently digested with RNase A/T1, an expected band of 205 nt was detected in all RNA samples (Fig. 3B). Similarly, when the antisense I/A ß_B-subunit riboprobe was hybridized with these RNA samples of equal amount, an expected fragment of 243 nt was protected (Fig. 3C). Similar to the results from semiquantitative RT-PCR, expression levels of the FSHß (high FSH: 0.67 ± 0.11; low FSH: 0.17 ± 0.11; p < 0.01) and I/A ß_B subunit (high FSH: 0.17 ± 0.03; low FSH: 0.09 ± 0.02; p < 0.05) genes differed between these two groups of boars. The FSHß and I/A ß_B-subunit transcripts were correlated (r = 0.68, p < 0.05).

View larger version (37K): [in this window] [in a new window]   FIG. 3. RPA. A) Schematic diagram of the assay and the expected protected fragments for the FSHß riboprobe. Expected fragments of 205 nt for FSHß (B), 243 nt for I/A ßB subunit (C), and 259 nt for ß-actin (D) were protected from all RNA samples. Animal identification is listed at the top; the six low FSH boars are on the left and the six high FSH boars are on the right. Marker represents the sizes from 100 to 500 nt. The yeast RNA controls, digested with RNase or without RNase (-RNase), are given in the last two lanes.

Comparison of FS and GnRH-R mRNAs between High and Low FSH Groups (RT-PCR)

With semiquantitative RT-PCR, expression levels of the FS (high FSH: 1.36 ± 0.14; low FSH: 1.02 ± 0.14; p > 0.10) and GnRH-R (high FSH: 0.87 ± 0.09; low FSH: 0.63 ± 0.08; p > 0.08) genes were not different between the high and low FSH groups. Similarly, the correlations between the transcripts of FSHß and FS (r = 0.43; p > 0.10) or GnRH-R genes (r = 0.46; p > 0.10) were not significant.

	DISCUSSION

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Both the semiquantitative RT-PCR and RPAs demonstrated significantly greater expression of the FSHß and I/A ß_B-subunit genes in anterior pituitary glands of boars with high FSH than in those with low FSH concentrations. No significant relationship between the FSHß and FS or GnRH-R mRNAs was detected in these same anterior pituitary glands. Previously, we reported that MS boars had markedly greater plasma FSH concentrations relative to WC boars [15, 16], and this difference was associated with significantly greater FSHß transcripts in pituitaries of MS compared to WC boars [17, 18]. Additionally, expression of the I/A ß_B-subunit gene in MS was about two times greater than in WC boar pituitaries [18]. The results of the current study support the conclusion that this difference in I/A ß_B gene expression is related to the difference in FSH synthesis and secretion rather than being an unrelated characteristic of the MS breed. By using crossbreds of MS and WC, large differences in FSH secretion were readily apparent among boars (Fig. 1) [20], with this difference in FSH being associated positively with differences in expression of the I/A ß_B-subunit gene (Fig. 2).

Our results do not support a strong relationship between FSHß and GnRH-R expression levels in boars and agree with earlier findings in female pigs, ewes, and cows that GnRH, at the physiological stages investigated, was not the major regulator of FSH secretion [28–31]. Even though LH secretion was stimulated by pulsatile administration of GnRH after hypothalamic-pituitary disconnection of pigs and ewes or nutritional restriction of cows, blood FSH concentrations in these same females were not affected by GnRH pulses. Furthermore, passive immunization of ovariectomized female pigs against GnRH decreased LH secretion rapidly within 3 h, whereas FSH secretion remained stable for 8 h and then decreased slowly during the next 8 days [32]. From these observations we predict that in boars, regulation of FSH by GnRH occurs at a secondary level and that activin is a primary regulator.

From observations obtained with perifusion of pituitary cells from male rats, Besecke et al. [33] determined an activin requirement for GnRH stimulation of FSHß gene expression. Additionally, they proposed that infrequent pulses of GnRH, as occur in males, stimulate FSHß expression without having an effect on FS expression. Present results in boars support this component of their model; however, they also observed that frequent pulses of GnRH stimulated FS expression resulting in decreased FSHß expression via FS inhibition of activin. Such differences in FS expression do not account for the diverse differences in FSHß expression or FSH secretion that we observe in boars (Figs. 1 and 3) [18].

In pigs, sex-specific differences in FSH secretion occur. During pubertal development in boars, FSH secretion increases in concert with testicular steroids, albeit the magnitude of change in FSH varied with the study [20, 34, 35]. In contrast, FSH secretion in females decreases during pubertal development [36, 37]. A number of possibilities may account for this gender difference, including a difference in inhibin secretion or sensitivity to inhibin feedback, or a difference in the relative roles of activin and GnRH in regulation of FSH synthesis. In females, GnRH may be the primary regulator rather than activin, or females may be more sensitive to negative feedback regulation of FSH by inhibin and/or steroids. Immunization against GnRH consistently reduced blood FSH concentrations of female pigs, but in boars the response has varied from no effect to inhibition ([38, 39]; K.L. Esbenshade, personal communication).

With differential-display PCR [22], a porcine calmodulin cDNA fragment of 290 bp was observed to be expressed in greater abundance in anterior pituitary RNA from WC than from MS boars (unpublished results). However, in the present study no expression differences were detected between boars classified as having high or low FSH concentrations when calmodulin-specific primers were used to amplify cDNA synthesized from pituitary RNA (Fig. 2). This indicates that the expression difference of the calmodulin gene between MS and WC boars is a breed difference independent of the elevated FSH concentrations detected in MS boars.

In summary, we demonstrated that expression of the I/A ß_B-subunit gene was positively associated with that of the FSHß gene in sexually mature boars, and the association between FSHß mRNA and plasma and pituitary FSH concentrations was also confirmed. In contrast, FSHß gene expression was not associated with FS or GnRH-R mRNAs in these crossbred boars. It remains to be determined why the boars with elevated plasma FSH concentrations are capable of expressing more I/A ß_B-subunit gene and whether significant differences of activin B can be detected in the pituitaries of boars with high FSH relative to those with low FSH.

	ACKNOWLEDGMENTS

 
The authors thank S. Hassler, R. Lee, D. Griess, and MARC Swine Operations Personnel for their skillful assistance and the USDA/NIDDK for FSH antisera, FSH for iodination, and FSH reference preparation.

	FOOTNOTES

 
¹ Names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the same by USDA implies no approval of the product to the exclusion of others that may also be suitable.

² Correspondence: J. Joe Ford, USDA, ARS, RLH U.S. Meat Animal Research Center, P.O. Box 166, State Spur 18D, Clay Center, NE 68933. FAX: 402 762 4382; ford{at}email.marc.usda.gov

³ Current address: Department of Pharmacology, University of Tennessee, Memphis, TN 38163.

Accepted: June 8, 1998.

Received: April 27, 1998.

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