Follicle-stimulating Hormone Beta Research Articles

Long-term exposure to continuous light inhibits precocity in European male sea bass ( Dicentrarchus labrax, L.): hormonal aspects

The effect of long-term exposure to continuous light on the hormonal aspects of the reproductive axis was investigated in juvenile male sea bass ( Dicentrarchus labrax, L.) during the first annual cycle. Four-month-old fish were exposed to a simulated natural photoperiod (NP) and a continuous light (24 h) regime (LL) under natural conditions of temperature (13.3–25.8 °C). A dot-blot technique was used to analyse gonadotropin (the common glycoprotein α, GPα; the follicle stimulating hormone beta, FSHβ; and the luteinizing hormone beta, LHβ, subunits) mRNA levels in the pituitary during the experiment. A homologous ELISA was used to determine pituitary sea bream gonadotropin-releasing hormone (sbGnRH) and LH and plasma LH levels; gonadal and plasma sex steroids concentrations were determined by specific immunoassays. LL significantly inhibited the expression of all three gonadotropins subunits in the pituitary. However, no significant differences on plasma LH levels were observed between NP and LL groups throughout the period of the experience. Long-term exposure to LL regime was extremely effective in inhibiting gonadal growth and hence precocious maturation as well as the accumulation of Testosterone (T) and 11-ketotestosterone (11-KT) in the gonads compared to the control group. 11-KT plasma levels remained low and unchanged in the LL group during the study. This work describe important alterations of the endocrine system, particularly at the pituitary–gonad axis provoked by exposure to continuous illumination and discusses the mechanism by which precocity in male sea bass is generated.

A single-chain tetradomain glycoprotein hormone analog elicits multiple hormone activities in vivo.

We previously demonstrated that genetically linking one or more of the glycoprotein hormone-specific beta subunit genes to the common alpha subunit resulted in single-chain analogues that were bioactive in vitro. The ability of such large structures to bind their cognate receptors with high affinity supported the hypothesis that extensive flexibility exists between the ligand and receptor to establish a functional complex. To further characterize the extent of this conformational flexibility, we engineered a single-chain analogue that consists of sequentially linked thyroid-stimulating hormone (TSH) beta, follicle-stimulating hormone (FSH) beta, and chorionic gonadotropin (CG) beta subunits to the alpha subunit and expressed this chimera in transfected CHO (Chinese hamster ovary) cells. Because the four subunits are genetically linked and expressed as a single-chain, this analogue presumably lacks significant native structural features of the individual heterodimers. However, it exhibited FSH, CG, and TSH activities in vitro. Here, we test whether this nonnative structure would be stable in vivo and thus biologically active. Using a variety of bioassay protocols, we demonstrate that the analogue elicits multihormone activities when injected in vivo. First, treatment with the analogue caused increases in ovarian and uterine weights and resulted in elevated serum estradiol. Second, the analogue-stimulated ovarian follicle growth and pharmacologically rescued in vivo FSH deficiency similar to recombinant human FSH or equine CG (eCG) as confirmed by induction of aromatase in the ovaries of FSHbeta knockout mice. Third, in a superovulation protocol, when primed with eCG, the analogue elicited a dose-dependent ovulatory response comparable with that by native heterodimeric human CG. Finally, the analogue-stimulated thyroxin production in hypothyroid mice similar to the pituitary-derived human TSH standard. Based on these data, we conclude that a single-chain tetradomain glycoprotein hormone analogue, despite its presumed altered conformation, is stable and biologically active in vivo. Our results establish the permissiveness and conformational plasticity with which the glycoprotein hormones are recognized in vivo by their target cell receptors.

Both SMAD2 and SMAD3 mediate activin-stimulated expression of the follicle-stimulating hormone beta subunit in mouse gonadotrope cells.

The activins are pleiotropic members of the TGFbeta superfamily. Within the anterior pituitary gland, activins stimulate FSH synthesis in an autocrine/paracrine fashion by stimulating transcription of the FSHbeta subunit gene. Here, the mechanisms mediating this effect were investigated in the murine gonadotrope cell line, LbetaT2. Recombinant activin A and activin B dose- and time-dependently stimulated endogenous FSHbeta mRNA expression. FSHbeta primary transcript and mRNA levels were increased within 30-60 min, but these effects were blocked by preincubation with the transcription inhibitor actinomycin-D, suggesting that the FSHbeta gene is a direct target of the activin signal transduction cascade. In other systems, activin signals are transduced through a heteromeric serine/threonine receptor complex, which includes the signaling activin type IB receptor [activin receptor-like kinase 4 (ALK4)]. Transfection of a constitutively active form of the receptor, ALK4T206D, stimulated FSHbeta mRNA levels. Overexpression of the inhibitory SMAD7 blocked this effect, as well as activin-stimulated FSHbeta transcription. Because SMAD7 functions by preventing access of SMAD2 and SMAD3 to ALK4, these data suggested that both activins and ALK4 require SMAD2 and/or SMAD3 to affect FSHbeta transcription. Consistent with this idea, activin A stimulated SMAD2 and SMAD3 phosphorylation and nuclear translocation within 5-10 min in LbetaT2 cells. Transient transfection of SMAD3, but not SMADs 1, 2, 4, 5, or 8, stimulated endogenous FSHbeta mRNA levels. The results of SMAD2 transfection studies were inconclusive, however, because of a persistent failure to overexpress the full-length SMAD2 protein specifically in LbetaT2 cells. To assess more directly roles for both SMAD2 and SMAD3 in activin-stimulated FSHbeta expression, RNA interference was used to decrease endogenous SMAD protein levels in LbetaT2 cells. Activin A- and ALK4T206D-stimulated transcription of the FSHbeta gene were significantly attenuated by the depletion of either SMAD2 or SMAD3. Collectively, these data suggest that activins use both SMAD2- and SMAD3-dependent mechanisms to stimulate FSHbeta transcription in mouse gonadotrope cells.

Orientation of Follicle-stimulating Hormone (FSH) Subunits Complexed with the FSH Receptor: β SUBUNIT TOWARD THE N TERMINUS OF EXODOMAIN AND α SUBUNIT TO EXOLOOP 3

Follicle-stimulating hormone (FSH) comprises an alpha subunit and a beta subunit, whereas the FSH receptor consists of two halves with distinct functions: the N-terminal extracellular exodomain and C-terminal membrane-associated endodomain. FSH initially binds to exodomain, and the resulting FSH/exodomain complex modulates the endodomain and generates signal. However, it has been difficult to determine which subunit of FSH contacts the exodomain or endodomain and in what orientation FSH interacts with them. To address these crucial issues, the receptor was Ala-scanned and the hormone subunits were probed with photoaffinity labeling with receptor peptides corresponding to the N-terminal region of the exodomain and exoloop 3 of the endodomain. Our results show that both regions of the receptors are important for hormone binding and signal generation. In addition, the FSH beta subunit is specifically labeled with the N-terminal peptide, whereas the alpha subunit is labeled with the exoloop 3 peptide. These contrasting results show that the FSH beta subunit is close to the N-terminal region and that the alpha subunit is projected toward exoloop 3 in the endodomain. The results raise the fundamental question whether the alpha subunit, common among the glycoprotein hormones, plays a major role in generating the hormone signal common to all glycoprotein hormones.

Nuclear factor Y and steroidogenic factor 1 physically and functionally interact to contribute to cell-specific expression of the mouse Follicle-stimulating hormone-beta gene.

FSH is a heterodimeric glycoprotein hormone secreted from the gonadotrope cell population of the anterior pituitary. Despite its crucial role in mammalian reproduction, very little is known about regulation of the FSH beta-subunit gene at the molecular level. In this report, we examine the basis for cell-specific expression of FSH beta using the mouse L beta T2 and alpha T3-1 gonadotrope-derived cell lines. Characterization of the hormonal content of L beta T2 and alpha T3-1 cells at the protein level classifies these cells as relatively mature and immature gonadotropes, respectively. We studied L beta T2 cell-specific expression of FSH beta using 398 bp of the mouse FSH beta regulatory region linked to a luciferase reporter gene in transient transfection assays. This mouse FSH beta promoter can direct reporter gene expression specifically to L beta T2 cells when compared with other pituitary- and non-pituitary-derived cell lines, including alpha T3-1 cells. Furthermore, it is induced by activin, and interruption of the autocrine activin loop in L beta T2 cells by the addition of follistatin reduces its expression. Truncation analysis indicates that several regions of the promoter are involved in this specificity and that these can be dissociated from activin regulation. We identify binding sites for the orphan nuclear receptor steroidogenic factor-1 and the heterotrimeric transcription factor nuclear factor Y and show that these elements functionally interact to regulate FSH beta gene expression in an L beta T2 cell-specific manner. Moreover, steroidogenic factor-1 and nuclear factor Y are shown to physically interact with each other. This study is the first to demonstrate the presence of basal FSH beta protein in L beta T2 cells and to identify specific elements within the FSH beta promoter that contribute to basal and cell-specific expression of the gene.

Cloning of the genes for the pituitary glycoprotein hormone alpha and follicle-stimulating hormone beta subunits in the Japanese crested ibis, Nipponia nippon.

We have isolated a part of the gene for the pituitary glycoprotein hormone common alpha subunit (PGHalpha) and the whole gene for the follicle-stimulating hormone beta subunit (FSHbeta) in the Japanese crested ibis (Nipponia nippon), a critically endangered bird species in East Asia. The nucleotide sequence of a part of the PGHalpha gene (5026 bp) contained three exons holding the whole coding and 3' untranslated regions, but lacked a 5' untranslated region. Its exon-intron structure was similar to that in mammals, but different from that in teleosts in the location of the second intron. For the FSHbeta gene, the nucleotide sequence of 7633 bp was assembled from two phage clones. The exon-intron structure of three exons and two introns was similar to that observed in mammals and teleosts. In the putative promoter region of the ibis FSHbeta gene, a progesterone responsive element (PRE)-like sequence and two AP-1 responsive element-like sequences reported in the ovine FSHbeta gene were not conserved in complete form. The increased number of ATTTA motifs in the putative 3' untranslated region in comparison with those in Japanese quail and chicken FSHbeta cDNA suggested that more rapid degradation of FSHbeta mRNA occurs in this species. Deduced amino acid sequences of the ibis PGHalpha and FSHbeta showed high similarities with those of the corresponding subunits of other avian species. This is the first report on the genomic sequences of the PGHalpha and FSHbeta in an avian species.

Analysis of the Cys82Arg mutation in follicle-stimulating hormone beta (FSHβ) using a novel FSH expression vector

ObjectiveTo determine the effect of the Cys82Arg FSHβ mutation from a patient with isolated FSH deficiency upon follicle-stimulating hormone (FSH) levels in vitro. DesignIn vitro analysis of the Cys82Arg mutation and comparison with the phenotype. SettingTertiary medical center setting. Patient(s)DNA sequence of the FSHβ gene and clinical description from a patient with isolated FSH deficiency. Intervention(s)Construction of a new vector containing the cDNAs for the α-subunit and β-subunit of FSH (pαFSHβ) followed by mutagenesis and transfection into Chinese hamster ovary cells. Main outcome measure(s)Immunoreactive and bioactive FSH levels from the CHO cellular media. Result(s)Although expression of both subunits was present, both immunoreactive and bioactive FSH levels were unmeasurable from cellular media containing the mutation versus wild type. Conclusion(s)The Cys82Arg mutation in a male with normal puberty and azoospermia results in profound deficiency of FSH in vitro, thereby confirming the molecular basis of hypogonadism in this patient and documenting the importance of the Cys residue at position 82 of the FSHβ subunit.

Follicle-stimulating Hormone Interacts with Exoloop 3 of the Receptor

The human follicle-stimulating hormone (FSH) receptor consists of two distinct domains of approximately 330 amino acids, the N-terminal extracellular exodomain and membrane-associated endodomain including three exoloops and seven transmembrane helices. The exodomain binds the hormone with high affinity, and the resulting hormone/exodomain complex modulates the endodomain where receptor activation occurs. It has been an enigma whether the hormone interacts with the endodomain. In a step to address the question, exoloop 3 of (580)KVPLITVSKAK(590) was examined by Ala scan, multiple substitution, assays for hormone binding, cAMP and inositol phosphate (IP) induction, and photoaffinity labeling. We present the evidence for the interaction of FSH and exoloop 3. A peptide mimic of exoloop 3 specifically and saturably photoaffinity-labels FSH alpha but not FSH beta. This is in contrast to photoaffinity labeling of FSH beta by the peptide mimic of the N-terminal region of the receptor. Leu(583) and Ile(584) are crucial for the interaction of FSH and exoloop 3. Substitutions of these two residues enhanced the hormone binding affinity. This is due to the loss of the original side chains but not the introduction of new side chains. The Leu(583) and Ile(584) side chains appear to project in opposite directions. Ile(584) appears to be so specific and to require flexibility and stereo specificity so that no other amino acids can fit into its place. Leu(583) is less specific. The improvement in hormone binding by substitutions was offset by the severe impairment of signal generation of cAMP and/or inositol phosphate. For example, the Phe or Tyr substitution of Leu(583) improved the hormone binding and cAMP induction but impaired IP induction. On the other hand, the substitutions for Ile(584) and Lys(590) abolished the cAMP and IP induction. Our results open a logical question whether Leu(583), Ile(584), and Lys(590) interact with the exodomain and/or the hormone. The answers will provide new insights into the mechanisms of hormone binding and signal generation.

Cloning and spatiotemporal expression of the follicle-stimulating hormone beta subunit complementary DNA in the African catfish (Clarias gariepinus).

The gene and cDNA encoding a putative follicle-stimulating hormone beta subunit (cfFSHbeta) from African catfish (Clarias gariepinus) were cloned. Similar to other FSHbeta genes, the cfFSHbeta gene consisted of three exons interrupted by two introns. The cfFSHbeta cDNA coded for a mature protein of 115 amino acids. The 12 cysteines that are required for the typical tertiary folding of glycoprotein hormone beta subunits were positionally conserved in cfFSHbeta. The cfFSHbeta mRNA expression was exclusively detected in the pituitary and was detectable before pubertal development was initiated. The cfFSHbeta transcript levels increased in particular during early stages of puberty and reached constantly high levels after the first appearance of spermatids in the testis. The cfFSHbeta mRNA-positive cells were localized in the proximal pars distalis. Castration of mature males caused elevated cfFSHbeta mRNA levels that were decreased by steroid replacement. Previous work indicated that the African catfish is an interesting model to study the regulation of gonadal functions because cfLH is able to activate both the catfish luteinizing hormone receptor (cfLH-R) and follicle-stimulating hormone receptor (cfFSH-R). Because cfFSH purification has failed so far, ongoing studies are directed toward the production of recombinant cfFSH. After all, the developmental and hormonal regulation of cfFSHbeta transcript levels opens the possibility for physiologically relevant actions of the putative cfFSH, next to the presumptive bifunctionally acting cfLH.

Transcriptional activation of the ovine follicle-stimulating hormone-beta gene by gonadotropin-releasing hormone involves multiple signal transduction pathways.

GnRH regulates gonadotrope cells through GnRH receptor activation of the PKC-, MAPK-, and calcium-activated signaling cascades. Due to the paucity of homologous model systems expressing FSHbeta, little is known about the specific mechanisms involved in transcriptional regulation of this gene by GnRH. Previous studies from our laboratory demonstrated that the gonadotrope-derived LbetaT2 cell line expresses FSHbeta mRNA. In the present study we characterized the mechanisms involved in GnRH regulation of the FSHbeta promoter using this cell model. Using transfection assays, we show that GnRH regulation of the ovine FSHbeta promoter involves at least two elements, present between -4152/-2878 and -2550/-1089 bp, in association with one or several elements within the proximal region of the promoter. Surprisingly, the two activating protein-1 sites previously shown to be involved in the FSHbeta response to GnRH in heterologous cells do not play a role in GnRH responsiveness in the gonadotrope cell model. Here we demonstrate that calcium influx itself is not sufficient to confer the response, but it is necessary for both 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and GnRH induction of the FSHbeta gene. Moreover, we show that GnRH regulation of FSHbeta gene expression is mediated by PKC and establish the presence of multiple PKC isozymes in LbetaT2 cells. Interestingly, GnRH and TPA induce activity of the FSHbeta promoter through different, although possibly overlapping, pools of PKC isoforms. This is further supported by the use of a MAPK inhibitor, which abolishes the induction of FSHbeta by GnRH, but not by TPA. In conclusion, we have demonstrated that calcium, PKC, and MAPK signaling systems are all involved in the induction of FSHbeta gene expression by GnRH in the LbetaT2 mouse gonadotrope cell model.

Effects of 4-nonylphenol on gene expression of pituitary hormones in juvenile Atlantic salmon ( Salmo salar)

Alkylphenols such as 4-nonylphenol (NP) are one of the wide variety of environmental chemicals reported to have estrogenic effects in both in vitro and in vivo studies. Induction of eggshell zona radiata proteins (Zrp) and vitellogenin (Vtg) mRNA and protein synthesis in the liver are widely used biomarkers for xenoestrogen exposure in fish. However, little work has been done to characterize the molecular effects of xenoestrogens on other potential target organs such as the pituitary. To evaluate pituitary effects and develop new potential biomarkers for xenoestrogens, the influences of NP and 17β-estradiol (E2) on the mRNA levels of pituitary gonadotropic hormone (GTH) beta subunits [leutinizing hormone beta (LHβ or GTH IIβ) and follicle stimulating hormone beta (FSHβ or GTH Iβ)], prolactin (PRL), growth hormone (GH) and the pituitary specific transcription factor (Pit-1) were investigated in individual male and female juvenile Atlantic salmon ( Salmo salar), 3 days after a single intraperitoneal (i.p.) injection. In one experiment, fish were injected with NP (125 mg/kg body weight (BW)) or E2 (5 mg/kg BW) and a semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) method was used to analyze LHβ and FSHβ mRNA levels. In the second experiment, fish were injected with three doses of NP (10, 50, 125 mg/kg BW) or a single dose of E2 (5 mg/kg BW) and Northern blot analysis was used to quantify LHβ, FSHβ, PRL, GH and Pit-1 mRNAs. Both NP (50 and 125 mg/kg BW) and E2 significantly induced LHβ mRNA levels ( P<0.01), but only in females. The highest dose of NP (125 mg/kg BW) significantly induced Pit-1 mRNA in males ( P<0.01). NP did not have significant effects on any of the other pituitary transcripts. NP induced LHβ mRNA synthesis in females by up to 6-fold and the changes appeared to correlate with the increases in hepatic Vtg and Zrp mRNA levels. The results show that LHβ mRNA assay in female juvenile salmonids may be used as a marker for pituitary effects of xenoestrogens. The data also suggest that NP may have the potential to perturb the regulation of LHβ gene expression by mimicking E2.

Effects of active immunization with inhibin alpha subunit on reproductive characteristics of turkey hens.

The hypothesis for the present study is that the active immunization of female turkeys with inhibin (INH) would neutralize endogenous INH, and increase levels of circulating follicle stimulating hormone (FSH) and the number of preovulatory follicles, and subsequently enhance egg production. Two experiments were conducted with female turkeys in their first (30 wk of age) and second (62 wk of age) laying cycles. Treatment groups included control turkeys immunized with keyhole limpet hemocyanine (KLH) and experimental turkeys immunized with recombinant turkey inhibin alpha conjugated to KLH (rtINH), vasoactive intestinal peptide (VIP) conjugated to KLH or rtINH+VIP. Egg production increased (P < 0.05) in VIP and rtINH+VIP immunized birds, but not in rtINH immunized hens in comparison with a control group. A similar number of ovarian follicles, arranged in the follicular hierarchy of laying hens, was observed in all experimental groups. However, there was a larger number of nongraded yellow follicles in rtINH-immunized (62.5%) and rtINH+VIP-immunized (73.5%) groups compared with that of controls, suggesting overstimulation by FSH. Anterior pituitary FSH beta subunit, LH beta subunit, and prolactin (PRL) mRNA contents were determined by Northern blot analysis and reverse transcriptase-polymerase chain reaction (RT-PCR) in laying hens at the end of the experimental period. Hens immunized with rtINH showed increased FSH beta subunit mRNA content, but no change in the content of LH beta subunit or PRL mRNA. Hens immunized with VIP or rtINH+VIP had significant increases in both pituitary LH beta subunit and FSH beta subunit mRNA contents, accompanied by a decline in PRL mRNA abundance. The magnitude of the increase in FSH beta subunit to INH immunoneutralization was greater in first-cycle hens than in second-cycle hens. These data suggest that active immunization of female turkeys with INH neutralizes endogenous INH and increases both circulating FSH and the number of preovulatory follicles. However, no significant increase in egg production was observed in INH-immunized hens. The data confirm previous reports that VIP immunoneutralization increases egg production in turkey hens and shows for the first time that it also increases FSH beta subunit and LH beta subunit gene expression.

Expression and localization of leptin receptor in the normal rat pituitary gland.

Leptin receptor (leptin-R) is a polypeptide consisting of a single transmembrane-spanning component. Recent studies performed by reverse transcriptase polymerase chain reaction (RT-PCR) have shown the production of leptin-R in various tissues including the pituitary, hypothalamus and reproductive organs. The localization of leptin-R protein in the pituitary gland, however, has not been extensively studied. This study deals with the expression of leptin-R in the normal rat pituitary gland, which was disclosed primarily in the plasma membrane fraction by immunoblotting and immunohistochemical staining methods. Double immunohistochemical staining revealed that the colocalization of leptin-R and anterior pituitary hormone expression was seen mainly in growth hormone (GH)-secreting cells (97.4 +/- 1.3%; GH-positive cells/leptin-R-positive cells), but in less than 1% of prolactin (PRL)-, adrenocorticotropic hormone (ACTH)-, thyroid-stimulating hormone-beta (TSH beta)- and follicle-stimulating hormone-beta (FSH beta)/luteinizing hormone-beta (LH beta)-positive cells. In contrast, leptin was localized most frequently in FSH beta/LH beta- and less frequently in TSH beta-positive cells. The above findings suggest that, in the rat anterior pituitary gland, there are paracrine relationships between leptin-producing cells and cells with leptin-R, which may regulate the function of GH cells.

The promoter for the ovine follicle-stimulating hormone-beta gene (FSHbeta) confers FSHbeta-like expression on luciferase in transgenic mice: regulatory studies in vivo and in vitro.

Transgenic mice harboring the ovine FSHbeta (oFSHbeta) promoter plus first intron (from -4741 to +759 bp) linked to a luciferase reporter gene (oFSHbetaLuc) were generated to determine whether this promoter can direct tissue-specific expression in vivo and serve as a model for studying hormonal regulation of the FSHbeta gene. Of six lines of transgenic mice analyzed, luciferase was detected uniquely in the pituitaries of five of them. Pituitary luciferase activity was decreased 51-99% by chronic GnRH treatment (Lupron depot). Orchidectomy caused a 2- to 8-fold increase, and ovariectomy caused a 2- to 27-fold increase in pituitary luciferase activity. Furthermore, pituitary luciferase expression was consistently higher on estrus than on diestrus (3- to 20-fold). These data strongly suggested that the transgene was expressed specifically in pituitary gonadotropes and regulated in the same way as the endogenous mouse FSHbeta gene. Using primary pituitary cell cultures prepared from these transgenic mice, basal luciferase expression was maximal on day 3 and then decreased by day 6 of culture, a pattern reflected by endogenous mouse FSH secretion. In these pituitary cultures, basal oFSHbetaLuc expression was decreased 61-82% by follistatin or 59-79% by inhibin. Similarly, mouse FSH secretion was decreased 71% by follistatin or 65% by inhibin. Progesterone inhibited oFSHbetaLuc expression by 44-51%, but it had no effect on endogenous mouse FSH secretion. Estradiol lowered FSH secretion by 21%, but did not decrease oFSHbetaLuc expression significantly. In conclusion, these data demonstrated the ability of the oFSHbeta promoter to direct expression of a reporter gene specifically to pituitary gonadotropes in transgenic mice. Studying oFSHbetaLuc expression in vivo and in cell cultures derived from pituitaries of these transgenic mice should prove useful for understanding many features of FSHbeta regulation.

Leptin and the pituitary.

In 1994, Zhang et al. of Rockefeller University in New York reported the first successful complementary DNA (cDNA) cloning of leptin by the positional cloning method. Leptin was identified as the gene of ob/ob mouse in genetic obesity syndromes. It has very strong food intake control, and body weight and energy expenditure. The name “leptin” derived from the Greek word leptos, meaning “thin.” We hereby review major advances leading to our current finding of leptin, leptin receptor and its structure, the outline of homozygote, and also influence of leptin in the pituitary.

A Radiation Map of the Short Arm of Porcine Chromosome 2

In recent years, maps of mammalian genomes have been acquiring increasingly higher resolution. Integration of maps of different types has become possible. As a tool in integrating maps of mammalian genomes of different types, high-resolution mapping with radiation-induced hybrids (RH) is used. Here, we present an RH6000 map of the short arm of porcine chromosome 2. The map contains 15 microsatellites and five genes (for parathyroid hormone, lactate dehydrogenase A, myogenic factor, follicle-stimulating hormone beta, and calpain I). The RH panel was obtained on the basis of a hybrid cell line bearing the single porcine chromosome 2 against the background of mink chromosomes. The mean frequency of preserving markers examined in the panel was 18.3%. Integration of four genes in the panel and a comparison of gene order in homeologous regions of human and porcine chromosomes are presented.

Candidate gene analysis for loci affecting litter size and ovulation rate in swine.

A candidate gene approach was used to determine whether specific loci explain responses in ovulation rate (OR) and number of fully formed (FF), live (NBA), stillborn, and mummified pigs at birth observed in two lines selected for ovulation rate and litter size compared with a randomly selected control line. Line IOL was selected for an index of OR and embryonic survival for eight generations, followed by eight generations of two-stage selection for OR and litter size. Line C was selected at random for 16 generations. Line COL, derived from line C at Generation 8, underwent eight generations of two-stage selection. Lines IOL and C differed in mean EBV by 6.1 ova and 4.7 FF, whereas lines COL and C differed by 2.2 ova and 2.9 FF. Pigs of Generation 7 of two-stage selection lines were genotyped for the retinol binding protein 4 (RBP4, n = 190) and epidermal growth factor (EGF, n = 189) loci, whereas pigs of Generations 7 and 8 were genotyped for the estrogen receptor (ESR, n = 523), prolactin receptor (PRLR, n = 524), follicle-stimulating hormone beta (FSHbeta, n = 520), and prostaglandin-endoperoxide synthase 2 (PTGS2, n = 523) loci. Based on chi-square analysis for homogeneity of genotypic frequencies, distributions for PRLR, FSHbeta, and PTGS2 were different among lines (P < 0.005). Differences in gene frequencies between IOL vs C and COL vs C were 0.33 +/- 0.25 and 0.16 +/- 0.26 for PRLR, 0.35 +/- 0.20 and 0.15 +/- 0.24 for FSHbeta, and 0.16 +/- 0.16 and 0.08 +/- 0.18 for PTGS2. Although these differences are consistent with a model of selection acting on these loci, estimates of additive and dominance effects at these loci did not differ from zero (P > 0.05), and several of them had signs inconsistent with the changes in allele frequencies. We were not able to find significant associations between the polymorphic markers and phenotypes studied; however, we cannot rule out that other genetic variation within these candidate genes has an effect on the traits studied.

Mutations in the follicle-stimulating hormone-beta (FSH beta) and FSH receptor genes in mice and humans.

Follicle-stimulating hormone (FSH), a dimeric glycoprotein synthesized in the anterior pituitary gland, is important for the production of sex steroids and gametes. FSH-beta (FSH beta) and FSH receptor (FSHR) knockout mice display impaired ovarian follicular development and infertility in females and small testes, oligospermia, and fertility in males. Humans with FSH beta gene mutations tend to have a more severe phenotype than those with FSHR gene mutations, although infertility and varying degrees of impaired sex steroid production occur in both types of mutations. Data from human and mouse mutations in the FSH beta and FSHR genes suggest that FSH is necessary for normal pubertal development and fertility in males and females.

Identification and characterization of a new allele for the beta subunit of follicle-stimulating hormone in Chinese pig breeds.

During evaluation of follicle-stimulating hormone-beta (FSHB) expression in anterior pituitary glands by an RNase protection assay (RPA), the expected fragment of 205 nucleotides at positions 759-963 was not detected in one boar that had moderate plasma and pituitary FSH concentrations. After subcloning and sequencing, mRNA from this boar lacked an 11-bp fragment (5'-CATTTGGAAAC-3') at nucleotide positions 807-817 of the 3'-untranslated region (3'-UTR, D allele). Wild-type FSHB (WT allele) was present in pituitary RNA and genomic DNA in both Meishan (MS) and White Composite (WC) pigs; whereas the D allele was present only in MS pigs (P < 0.01; 5/6 MS vs. 0/6 WC). Also, we found the D allele in five other Chinese breeds but absent in ten American Landrace, 11 Yorkshire and 17 Berkshire pigs. Additionally, the D allele had one silent nucleotide change in the coding region plus six, single nucleotide changes in the 3'-UTR.

LbetaT2 gonadotroph cells secrete follicle stimulating hormone (FSH) in response to active A.

Secretion of luteinizing hormone in response to gonadotropin releasing hormone (GnRH) has been described in the recently developed LbetaT2 gonadotroph cell line. We evaluated the expression of follicle stimulating hormone (FSH)beta mRNA and secretion of FSH from LbetaT2 cells in response to GnRH and activin A. LbetaT2 cells were treated with activin A in doses from 0 to 50 ng/ml, with or without a daily 10 nM GnRH pulse, or with GnRH alone. FSH secretion was stimulated over 6-fold by concomitant GnRH and activin A in a dose-responsive fashion at 72 h of treatment. FSHbeta mRNA was detectable by ribonuclease protection assay only in cells treated with activin A with or without GnRH. The demonstration of FSHbeta gene expression in LbetaT2 cells further validates these cells as mature, differentiated gonadotrophs and as an important tool for the study of gonadotroph physiology.