The developmental genetics of Hirschsprung's disease.

Hirschsprung's disease (HSCR), also known as aganglionic megacolon, derives from a congenital malformation of the enteric nervous system (ENS). It displays an incidence of 1 in 5000 live births with a 4:1 male to female sex ratio. Clinical signs include severe constipation and distended bowel due to a non-motile colon. If left untreated, aganglionic megacolon is lethal. This severe congenital condition is caused by the absence of colonic neural ganglia and thus lack of intrinsic innervation of the colon due in turn to improper colonization of the developing intestines by ENS progenitor cells. These progenitor cells are derived from a transient stem cell population called neural crest cells (NCC). The genetics of HSCR is complex and can involve mutations in multiple genes. However, it is estimated that mutations in known genes account for less than half of the cases of HSCR observed clinically. The male sex bias is currently unexplained. The objective of this review is to provide an overview of the pathophysiology and genetics of HSCR, within the context of our current knowledge of NCC development, sex chromosome genetics and laboratory models.

Use of a biological peptide pump to study chronic peptide hormone action in transgenic mice. Direct and indirect effects of angiotensin II on the heart.

Angiotensin II is a peptide hormone regulator of blood pressure and fluid balance in mammals. Evidence obtained largely in vitro has also suggested that angiotensin II has growth-promoting effects and that it might thereby contribute to such pathological phenomena as cardiac hypertrophy, a major risk factor for cardiovascular mortality. It has been difficult to test for the direct growth-promoting effects of angiotensin II in vivo, however, because of the generalized effects of the peptide on hemodynamics. To overcome this limitation and to test for cardiac-specific functions of angiotensin II, we generated transgenic mice expressing an angiotensin II-producing fusion protein exclusively in cardiac myocytes. Our findings are the first to distinguish between local and systemic effects of angiotensin II on the heart and introduce a novel technique for studying tissue-specific peptide function.

Development and application of a biological peptide pump for the study of the in vivo actions of angiotensin peptides.

In the past few years, a great deal of interest has been focused on the possibility that angiotensin peptides could have direct effects on target tissues independent of their hemodynamic effects. In addition, there has been much speculation on the potential biological roles of angiotensin peptides other than angiotensin II. Unfortunately, a direct test of these possibilities in whole animals has been difficult due to limitations in existing biological systems. In this review, we describe the characteristics of an engineered protein capable of directing the production of a wide variety of peptides to specific tissues and cell types in transgenic animals and discuss its potential applications.

Genetic manipulation of sex differentiation and phenotype in domestic animals.

In mammals, a gene based sex determination system ensures that approximately 50% of offspring will be of the male sex and 50% will be of the female sex. In domestic animal production systems, this ratio is not always ideal. Recent advances in our understanding of the molecular biology of sex determination and differentiation, as well as in the control of gene expression and the direct modification of animal genomes, allows us to consider methods for the direct genetic manipulation of sexual phenotype.

Molecular characterization of bovine prostaglandin G/H synthase-2 and regulation in uterine stromal cells.

Prostaglandin G/H synthase (PGHS) is a key rate-limiting enzyme in the prostaglandin biosynthetic pathway, and prostaglandins play a central role in the control of the reproductive cycle. The objectives of this study were to clone and characterize the primary structure of bovine PGHS-2 and to study its regulation in uterine stromal cells in vitro. The bovine PGHS-2 cDNA was cloned by a combination of reverse transcription-polymerase chain reaction and cDNA library screening. Results showed that the complete bovine PGHS-2 cDNA is composed of a 5'-untranslated region of 128 bp, an open reading frame of 1815 bp, and a 3'-untranslated region of 1565 bp containing multiple repeats (n = 11) of the Shaw-Kamen sequence 5'-ATTTA-3'. The open reading frame encodes a 604-amino acid protein that is 86-97% identical to other mammalian PGHS-2 homologs. The regulation of PGHS-2 mRNA and protein was studied in primary cultures of bovine uterine stromal cells stimulated with phorbol 12-myristate 13-acetate (PMA; 100 nM). Northern and Western blot analyses reveal a marked induction in PGHS-2 transcript (4.0 kilobases) and protein (M(r) = 72 000) after 3-12 h of PMA stimulation (P < 0.05). However, this induction was transient in nature as levels of PGHS-2 mRNA and protein returned to basal levels after 24 h of PMA stimulation. In contrast, PMA had no effect on levels of PGHS-1 (P > 0.05). The PMA-dependent induction of PGHS-2 was associated with a significant increase in prostaglandin E2 secretion in the culture media (P < 0.05). To study promoter activity of the 5'-flanking DNA region of the bovine PGHS-2 gene, the genomic fragment -1574/-2 (+1 = transcription start site), as well as a series of 5'-deletion mutants, were fused upstream of the firefly luciferase gene and transiently transfected into primary cultures of bovine uterine stromal cells. Results showed that a first promoter region located between -1574 and -492 and a second region between -88 and -39 appear to play important roles in PMA-dependent regulation of PGHS-2 promoter activity in bovine uterine cells. Thus, this study characterizes for the first time the structure of the bovine PGHS-2 transcript and the deduced amino acid sequence of its encoded protein and establishes an in vitro model to study the regulation of PGHS-2 gene expression in bovine uterine tissue.

Expression and regulation of transcripts encoding two members of the NR5A nuclear receptor subfamily of orphan nuclear receptors, steroidogenic factor-1 and NR5A2, in equine ovarian cells during the ovulatory process.

Steroidogenic factor-1 (SF-1, NR5A1a) is a member of the NR5A nuclear receptor subfamily and has been implicated as a key transcriptional regulator of all ovarian steroidogenic genes in vitro. To establish links between the expression of SF-1 and that of the steroidogenic genes in vivo, the objectives of this study were to clone equine SF-1 and examine the regulation of its messenger RNA (mRNA) in follicular cells during human CG (hCG)-induced ovulation. The equine SF-1 primary transcript was cloned by a combination of RT-PCR techniques. Results showed that the transcript was composed of a 5'-untranslated region (UTR) of 161 bp, an open reading frame (ORF) of 1386 bp that encodes a highly-conserved 461-amino acid protein, and a 3'-UTR of 518 bp. The cloning of SF-1 also led to the unexpected and serendipitous isolation of the highly-related orphan nuclear receptor NR5A2, which was shown to include a 5'-UTR of 243 bp, an ORF of 1488 bp, and a 3'-UTR of 1358 bp. The NR5A2 ORF encodes a 495-amino acid protein that is 60% identical to SF-1, including 99%-similar DNA-binding domains. Northern blot analysis revealed that SF-1 and NR5A2 were expressed in all major steroidogenic tissues, with the exception that NR5A2 was not present in the adrenal. Interestingly, NR5A2 was found to be, by far, the major NR5A subfamily member expressed in the preovulatory follicle and the corpus luteum. Using a semiquantitative RT-PCR/Southern blotting approach, the regulation of SF-1 and NR5A2 mRNAs in vivo was studied in equine follicular cells obtained from preovulatory follicles isolated between 0 and 39 h post hCG. Results showed that the theca interna was the predominant site of SF-1 mRNA expression in the follicle, and that hCG caused a significant decrease in SF-1 levels between 12-39 h in theca interna and between 24-39 h post hCG in granulosa cells (P < 0.05). In contrast, the granulosa cell layer was the predominant, if not the sole, site of NR5A2 mRNA expression in the follicle. Importantly, NR5A2 was much more highly expressed in granulosa cells than SF-1. The administration of hCG caused a significant decrease in NR5A2 transcripts in granulosa cells at 30, 36, and 39 h post hCG (P < 0.05). Thus, this study is the first to report the concomitant regulation of SF-1 in theca interna and granulosa cells throughout the ovulation/luteinization process, and to demonstrate the novel expression and hormonal regulation of NR5A2 in ovarian cells. Based on the marked expression of NR5A2 in equine granulosa and luteal cells and on mounting evidence of a functional redundancy between SF-1 and NR5A2 in other species, it is proposed that NR5A2 may play a key role in the regulation of gonadal steroidogenic gene expression.

Contribution of circulating renin to local synthesis of angiotensin peptides in the heart.

The activity of a local cardiac renin-angiotensin system (RAS) has long been suspected in the promotion of cardiac pathologies including hypertrophy, ischemia, and infarction. All of the components of the RAS cascade have been demonstrated to be synthesized within the heart with the possible exception of the first enzyme in the cascade, renin. In the current study, we provide direct evidence that circulating renin can contribute to cardiac-specific synthesis of angiotensin peptides. Furthermore, we demonstrate this effect is independent of blood pressure and that in animals of comparable blood pressure, elevated circulating renin significantly enhances cardiac fibrosis. These results may serve to explain some of the cardiac pathologies associated with the RAS.

Vacuolar system-associated protein-60: a protein characterized from bovine granulosa and luteal cells that is associated with intracellular vesicles and related to human 80K-H and murine beta-glucosidase II.

It has been suggested that proteins of molecular size 56-58 kDa play an important role in bovine ovarian follicular development and oocyte maturation. A polyclonal antibody was raised against a 56- to 58-kDa protein band purified from bovine granulosa cells and was used to screen granulosa or luteal cell cDNA expression libraries. This work resulted in the identification of a cDNA encoding for a protein of 60.1 kDa with a signal peptide of 13 residues. The bovine 60.1-kDa protein shared an overall 86.7% and 81.8% identity with, respectively, the human 80K-H protein and the mouse putative beta subunit of glucosidase II (beta-GII), and was named vacuolar system-associated protein-60 (VASAP-60). Marked differences in sequence identity were noted in a putative molecular adapter domain containing a tandem D and E amino acid stretch flanked by proline-rich sequences presenting the minimal PXXP SH3 motif. VASAP-60 was shown to be unglycosylated using endoglycosidase H treatment and was found mainly in a cellular membrane fraction of bovine corpus luteum. VASAP-60 was localized in a rat hepatic Golgi/endosome fraction and in wheat germ agglutinin (WGA) affinity chromatographic eluates, thereby suggesting the presence of interactions with membrane glycoproteins. A polyclonal antibody was raised against the putative adapter domain of the recombinant VASAP-60; this was shown to recognize a major 88-kDa and two minor 58-kDa and 50-kDa proteins, suggesting that the major 88-kDa protein band represents the complete VASAP-60 protein whereas the 58-kDa and the 50-kDa bands represent its proteolytic fragments. Northern blot analysis demonstrated the presence of a single 2.3-kilobase transcript in all the bovine tissues analyzed with variation in the steady state level between tissues. Immunohistochemical observations showed that VASAP-60 was widely distributed in bovine tissues and was localized in pericytoplasmic and perinuclear membranes. In epithelial cells, the staining presented a basolateral or apical polarity associated with intracellular vacuoles. In conclusion, we have characterized a novel acidic membrane protein, associated with organelles of the vacuolar system, that is widely and histospecifically expressed in bovine tissues. VASAP-60 represents either the bovine ortholog or a new family member of the previously characterized human 80K-H and murine beta-GII proteins. Our results suggest that VASAP-60 presents characteristics of a molecular adaptor protein with functions in membrane-trafficking events.

Formation of corneal endothelium is essential for anterior segment development - a transgenic mouse model of anterior segment dysgenesis.

The anterior segment of the vertebrate eye is constructed by proper spatial development of cells derived from the surface ectoderm, which become corneal epithelium and lens, neuroectoderm (posterior iris and ciliary body) and cranial neural crest (corneal stroma, corneal endothelium and anterior iris). Although coordinated interactions between these different cell types are presumed to be essential for proper spatial positioning and differentiation, the requisite intercellular signals remain undefined. We have generated transgenic mice that express either transforming growth factor (alpha) (TGF(alpha)) or epidermal growth factor (EGF) in the ocular lens using the mouse (alpha)A-crystallin promoter. Expression of either growth factor alters the normal developmental fate of the innermost corneal mesenchymal cells so that these cells often fail to differentiate into corneal endothelial cells. Both sets of transgenic mice subsequently manifest multiple anterior segment defects, including attachment of the iris and lens to the cornea, a reduction in the thickness of the corneal epithelium, corneal opacity, and modest disorganization in the corneal stroma. Our data suggest that formation of a corneal endothelium during early ocular morphogenesis is required to prevent attachment of the lens and iris to the corneal stroma, therefore permitting the normal formation of the anterior segment.

The delayed activation of the prostaglandin G/H synthase-2 promoter in bovine granulosa cells is associated with down-regulation of truncated upstream stimulatory factor-2.

To elucidate the molecular mechanisms involved in the delayed induction of PGHS-2 in species with a long ovulatory process, a 1. 6-kilobase fragment of the bovine PGHS-2 promoter was isolated, and its activity was characterized in primary cultures of bovine granulosa cells. Promoter activity assays performed with a series of deletion mutants revealed that the promoter region from -149 to -2 (+1 = transcription start site) confers full-length promoter activity in response to forskolin (10 microM). Four consensus cis-elements were identified within this region, including an E-box, ATF/CRE, C/EBP, and AP2 site. Site-directed mutagenesis showed that the E-box was required for PGHS-2 promoter activity, that disruption of the C/EBP element decreased forskolin inducible activity by 29%, whereas point mutation within the ATF/CRE and AP2 element had no inhibitory effect. Electrophoretic mobility shift assays (EMSAs) performed with the -149/-2 fragment and granulosa cell nuclear extracts obtained before (0 h) and after (18 and 20 h) human chorionic gonadotropin (hCG) revealed the regulation of multiple DNA-protein complexes. The 0-h extract generated four complexes at the E-box, whereas only one complex was produced at this site with the 18-h extract. Supershift EMSAs identified that upstream stimulatory factor-1 and -2 (USF-1 and -2) were part of these complexes. Interestingly, the presence of the amino-terminal truncated USF-2, which lacks the transcription activation domain, was detected in the 0-h extract, but not in extracts prepared post-hCG. Supershift EMSAs also indicated high levels of C/EBPbeta binding to its cis-element in the 0-h extract, which contrasts with results previously reported in rats. Thus, high levels of amino-terminal truncated USF-2 and C/EBPbeta in bovine granulosa cells prior to hCG treatment could repress gene expression, and be involved in the delayed induction of PGHS-2 in species with a long ovulatory process.

High expression of bovine alpha glutathione S-transferase (GSTA1, GSTA2) subunits is mainly associated with steroidogenically active cells and regulated by gonadotropins in bovine ovarian follicles.

We have previously shown that a major group of 28-30 kDa proteins decreases after the LH surge in bovine granulosa cells (GC). In the present study, we have characterized two proteins in this group in search of factors that may intervene in folliculogenesis and oocyte maturation. Polyclonal antibodies raised against 28 kDa or 29 kDa bovine GC proteins were used to screen a complementary DNA (cDNA) expression library. This resulted in the characterization of two isoenzyme subunits for alpha class glutathione S-transferase, named bGSTA1 and bGSTA2. Both bGSTA1 (25.4 kDa, pI 8.9; 791 bp cDNA; GenBank Accession No. BTU49179) and bGSTA2 (25.6 kDa, pI 7.2; 959 bp cDNA; GenBank Accession No. AF027386) have 222 amino acids. The deduced amino acid sequences were compared and showed 82% (bGSTA1) and 74% (bGSTA2) identity to human GSTA1, whereas bGSTA1 and bGSTA2 are 81% identical to each other. The bGSTA2 represents a novel GSTA subunit because it harbors a specific 16 amino acid sequence not found in any other species and GST classes. Northern blots showed that bGSTA1 and bGSTA2 are coexpressed and are tissue specific with single transcripts of 1.2 kb and 1.4 kb, respectively for bGSTA1 and bGSTA2. The messenger RNA (mRNA) were detected in GC, corpus luteum, adrenal gland, testis, liver, lung, thyroid, kidney and cotyledon, and the relative abundance of their mRNA varied. Ratios of bGSTA1/bGSTA2 mRNA vary between tisssues, indicating that expression of these genes is controlled differently. Immunohistochemistry observations revealed that expression of GSTA is cell specific, being associated with GC and theca cells, small luteal cells, Leydig cells, hepatocytes, adrenal cortex, specific chromaffin cells in the adrenal medulla, renal proximal convoluted tubular cells, and Clara cells in the bronchioles. Studies in vivo showed that levels of mRNA for bGSTA1 were elevated in follicular wall of preovulatory follicles before hCG treatment, but decreased by 77% 12 h after hCG injection. However, in FSH stimulated preovulatory follicles, the decrease in mRNA for both GSTAs was only 21% at 24 h following hCG injection. We concluded that bGSTA1 and bGSTA2 expression is tissue- and cell-specific, is associated with steroidogenically active cells, and is hormonally regulated by gonadotropins in the bovine ovarian follicle.

In vivo enzymatic assay reveals catalytic activity of the human renin precursor in tissues.

The aspartyl protease renin is secreted into the circulation of mammals in 2 forms: the proteolytically processed active form of the enzyme and the precursor form, prorenin. Prorenin has no detectable enzymatic activity in the circulation, but it is the exclusive form of the enzyme produced by several tissues that also produce the other components of the renin enzymatic cascade (renin-angiotensin system). To test whether prorenin might be enzymatically active in these tissues, transgenic mice expressing the human renin substrate (angiotensinogen) exclusively in the pituitary gland were mated to mice expressing either active human renin or prorenin in the same tissue. Measurement of in vivo product formation in pituitary glands of double-transgenic mice revealed that human prorenin was enzymatically active, and Western blot analysis demonstrated that this prorenin was in the precursor form with its prosegment attached. This in vivo enzymatic assay demonstrates for the first time that human prorenin can be activated within tissues by nonproteolytic means, where it could contribute to the activity of a localized renin-angiotensin system.

Porcine steroidogenic factor-1 gene (pSF-1) expression and analysis of embryonic pig gonads during sexual differentiation.

The porcine steroidogenic factor-1 gene (pSF-1) was cloned using a combination of genomic and RT-PCR based cloning methods. pSF-1 consists of an open reading frame of 1383 nt corresponding to a deduced amino acid sequence of 461 aa, similar to bovine and human SF-1. Sequence homologies between pSF-1 and human, bovine and mouse molecules indicate strong evolutionary conservation at both the nt and aa levels. Northern analysis of pSF-1 expression in adult steroidogenic tissues correlated with porcine steroidogenic acute regulatory protein gene (pStAR) and porcine side chain cleavage (pP450scc) gene expression. Notably, pSF-1 expression was readily detected in neonatal testes, absent at 3 weeks of age, and again readily detected at 3 months and in adult testes. pSF-1 expression was weak but detectable in placental tissues at various times of gestation, and was correlated with pStAR and pP450scc expression, indicating classical steroidogenesis in this organ. In developing gonads from 6-12 weeks of gestation, i.e. during the time of sex differentiation in the pig, Northern analysis demonstrated increasing expression of PSF-1 in fetal testes and no expression in ovaries. This expression pattern was paralleled for pStAR, pP450scc, and porcine Müllerian inhibitory substance (pMIS), consistent with pSF-1 involvement in both steroid and protein hormone secretions of the developing testes during sex differentiation. Porcine SRY HMG-box related gene-9 (pSOX-9) expression also paralleled that of pSF-1 in developing testes. In contrast, DSS-AHC critical region on the X chromosome, gene 1 (pDAX-1) was expressed predominantly in the developing ovaries, indicating a possible reciprocal regulation of pSF-1 and pDAX-1 genes in developing pig testes and ovaries.

Bovine activin receptor type IIB messenger ribonucleic acid displays alternative splicing involving a sequence homologous to Src-homology 3 domain binding sites.

Activins are implicated in a variety of biological effects, particularly in reproductive processes such as embryonic development and folliculogenesis. Breakthroughs in the elucidation of the activin signal transduction mechanism were achieved with the characterization of the activin receptors, and the recent identification of cytoplasmic factors apparently involved in the signaling process. The present studies were undertaken to further analyze the activin signaling pathway. The complementary DNA coding for the bovine activin receptor type IIB (bActRIIB) was amplified by RT-PCR from corpus luteum and pituitary RNA, and cloned to characterize its role in activin signal transduction. Two complementary DNA isoforms (bActRIIB2 and bActRIIB5) were detected, coding for 512 amino acids and 498 amino acids, respectively. The shortest isoform lacked a sequence encoding a 14-amino acid stretch very rich in proline residues, located between the transmembrane region and the intracellular kinase domain. Intron sequencing and ribonuclease protection assay demonstrated that alternative splicing is responsible for the generation of these bActRIIB isoforms. This alternative splicing event is unique in that it has not been observed in other species, including the mouse, in which extensive alternative splicing of the ActRIIB messenger RNA is described. Comparison of this alternative sequence with other known proline-rich sequences showed that it has characteristics of a Src-homology 3 domain (SH3) binding site. Coprecipitation experiments have identified two proteins of 69 kDa and 71 kDa from an uterine endometrial cell line, specifically interacting with the short bActRIIB alternative proline-rich sequence. These results suggest that bActRIIB could have a protein-protein interaction, through its putative SH3 binding site, with at least two intracellular SH3-containing proteins.

Bovine thyrotropin receptor cDNA is characterized by full-length and truncated transcripts.

The complete coding sequence for the bovine thyrotropin (TSH) receptor was derived using a modified PCR cloning strategy. The bovine thyrotropin receptor conforms to the pattern of receptor interacting with membrane-bound G-protein already established in other species for TSH and gonadotropins receptors. The cDNA for the bovine TSH receptor consists of an open reading frame 2289 nucleotides in length, corresponding to a protein of 763 amino acids (estimated molecular mass of 86.4 kDa) which includes a 20 amino acid putative leading signal peptide. The receptor consists of a large NH2-terminal extracellular membrane domain of 417 amino acids with 5 potential N-linked glycosylation sites, a transmembrane domain (265 amino acids) consisting of 7 putative membrane alpha-helix spanning segments, and an intracytoplasmic COOH-terminal domain (82 amino acids). The bovine TSH receptor is one amino acid less than the corresponding sequence in dog, human, rat and mouse. Cysteine residues (n = 22) were conserved when compared with other TSH receptors. Three potential phosphorylation sites were found in the transmembrane domain and the COOH-terminal domain. As with other members of this receptor family, alternative splicing was observed. A transcribed but truncated TSH receptor of 1769 nucleotides was demonstrated, lacking half of the V segment of the transmembrane domain up to the COOH-terminal domain of the full length TSH receptor. Additionally, alternative transcriptional start sites were observed. Northern blot analysis using a probe (1170 bp) spanning part of the extracellular domain up to the first loop of the transmembrane domain showed specific expression in the bovine thyroid gland with major transcripts of 9.3 and 4.3 kb, and a minor transcript of 3.8 kb being detected.

Porcine and bovine steroidogenic acute regulatory protein (StAR) gene expression during gestation.

We have generated complete complementary DNA (cDNA) sequences for the porcine steroidogenic acute regulatory protein (StAR) gene, using a combination of genomic PCR amplification and reverse transcription-PCR amplification of pig ovarian cDNA. Porcine StAR cDNA consists of 855 bp and shares 90.2%, 87.3%, 84.3%, and 83.9% homologies with bovine, human, mouse, and rat StAR cDNA at the nucleotide level, and 89.1%, 88.8%, 86.7%, and 86.3% homologies with bovine, human, mouse, and rat StAR protein at the deduced amino acid level. Northern analysis of porcine StAR showed that it is expressed in adult and fetal steroidogenic tissues, including adult testes and ovaries and adult adrenal glands as well as steroidogenic tissues of pregnancy, including developing fetal testes, corpus luteum, and pregnancy, but not the fetal ovary. Major hybridizing bands of 1.8 and 1.1 kilobases were demonstrated. In contrast to human StAR, porcine StAR was not expressed in adult or fetal kidneys. Expression of porcine StAR by the pig placenta is in contrast to human StAR, which is not expressed by the human placenta. Northern analysis of bovine cotyledons using a homologous probe for bovine StAR showed that StAR is also expressed by the placenta in the bovine animal. With respect to placental expression of StAR, variations may exist among mammalian species.

Follicular 3 beta-hydroxysteroid dehydrogenase and cytochromes P450 17 alpha-hydroxylase and aromatase messenger ribonucleic acids in cattle undergoing superovulation.

In this study, we tested the hypothesis that there is altered abundance of transcripts of genes coding for the enzymes cytochrome P450 17 alpha-hydroxylase (P450(17 alpha)), cytochrome P450 aromatase (P450arom), and 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) in follicles of cattle hyperstimulated with eCG compared to FSH. Treatments were initiated on Day 10 of the cycle, and all cows received prostaglandin (PG) on Day 12. In experiment 1, blood samples were taken to determine plasma progesterone and estradiol concentrations during ovarian stimulation. In experiment 2, both ovaries were removed from stimulated cows by colpotomy before (n = 4 cows/treatment) and at 12 (n = 3/treatment) and 24 h (n = 3/treatment) after PG injection, and from nonstimulated controls (n = 4) 72 h after PG. The preovulatory follicle from nonstimulated heifers, and all follicles greater than 3 mm in diameter from superovulated heifers, were isolated and classified as small (3-5 mm), medium (6-9 mm), or large (> 9 mm). Steady-state levels of RNA for 3 beta-HSD, P450(17 alpha), and P450arom genes were determined by Northern analysis in the individual follicles. In experiment 1, stimulation with eCG significantly (p < 0.01) increased plasma progesterone concentrations compared to FSH-stimulated and nonstimulated controls, and increased (p < 0.05) plasma estradiol concentrations compared to FSH-stimulated controls. Stimulation with FSH did not alter progesterone concentrations, but significantly increased plasma estradiol concentrations compared to those of controls. In experiment 2, the number of large follicles increased significantly with time (p < 0.01), but there were no differences between eCG and FSH treatments in size distribution of follicles (p > 0.05). Relative abundance of P450(17 alpha) message (per 20 micrograms RNA) was significantly higher in large and small follicles (p < 0.05) in eCG-treated compared to FSH-treated heifers after PG injection. Analysis within this period revealed significant treatment effects at 12 h but not 24 h after PG injection. The bovine P450arom cDNA hybridized to 3 transcripts: a 6.5-kilobase (kb) polyadenylated transcript, and non-polyadenylated messages of 3.4 and 1.8 kilobases (kb), all of which hybridized with an oligonucleotide probe specific for the heme-binding region. In medium and small follicles, the 6.5-kb and 3.4-kb transcripts were present in similar quantities, and the 1.8-kb transcript was 25% less abundant. In large follicles recovered after luteolysis, the 3.4 and 1.8-kb transcripts were 3- to 4-fold more abundant in eCG-treated compared with FSH-treated and nonstimulated animals (p < 0.05). There were no significant differences between eCG and FSH treatments on steady-state 3 beta-HSD mRNA levels. Levels of 3 beta-HSD and P450(17 alpha) mRNA in large follicles in hyperstimulated heifers were not different from those in preovulatory follicles in nonstimulated cows. We conclude that hyperstimulation with eCG results in greater stimulation of follicular P450(17 alpha) message abundance compared to hyperstimulation with FSH, and that this may contribute to increased follicular estradiol secretion.

Porcine SRY gene locus and genital ridge expression.

Porcine SRY gene locus was cloned through use of a strategy of anchored polymerase chain reaction (PCR) amplification from a male pig genomic DNA size-selected library constructed in a plasmid vector as well as 3' reverse transcription (RT)-PCR amplification of porcine genital ridge SRY transcripts. In total, 1664 bp of genomic DNA and 106 bp of 3' cDNA are presented. The open reading frame of porcine SRY consists of 624 bp representing 208 amino acids (aa) with a centrally located HMG box domain of 79 aa, an amino-terminal region of 59 aa, and a carboxy terminal of 70 aa. Structurally, porcine SRY resembles human and bovine SRY more closely than it does mouse Sry, and it lacks the carboxy-terminal activation domain seen in the mouse Sry molecule. Similar to human and bovine testicular SRY transcripts, the porcine SRY genital ridge transcript has a relatively short 3' untranslated region (UTR), in contrast to the extended UTR of the mouse genital ridge Sry transcript. The porcine SRY gene is expressed within the cells of the genital ridge of the developing male pig embryo between Days 21 and 26 (e21-e26) of gestation, during which time the primitive gonads are bipotential, but not on Day e31, by which time male testis determination is histologically evident.