OB-cadherin cloning and expression in a model of wound repair in horses.

REASON FOR PERFORMING STUDY: Horses suffer from a debilitating impediment in repairing wounds located on the lower limb that leads to the development of a fibroproliferative disorder (exuberant granulation tissue). This condition is a source of wastage since it often forces retirement from competition. Treatments that resolve or prevent this condition are still lacking, maybe due to deficient knowledge of the underlying molecular mechanisms. Fibroblast-to-myofibroblast conversion is an essential step allowing contraction during wound repair and is accompanied by an increase in OB-cadherin expression. OBJECTIVES: To clone equine cadherin-11 (CDH11) cDNA and to study its spatiotemporal expression profile during the repair of body and limb wounds, thereby contributing to a better understanding of the repair process. METHODS: Cloning was by a PCR technique. Expression was studied in intact skin and in 1, 2, 3, 4 and 6-week-old wounds of the body and limb. Temporal CDH11 gene expression was determined by RT-PCR while OB-cadherin protein expression was mapped immunohistochemically. RESULTS: Equine CDH11 is a highly conserved gene and protein. mRNA was not expressed in equine skin whereas the wound repair process was characterised by a significantly higher expression in the thorax than in limb samples. mRNA expression pattern was paralleled by protein data as confirmed by immunohistochemistry. CONCLUSIONS: The data suggest that deficient OB-cadherin expression in the first phases of wound repair contributes to the excessive proliferative response seen in horse limb wounds. POTENTIAL RELEVANCE: Future studies should verify the quantitative, temporal expression of this protein in order to provide the basis for targeted therapies that might prevent the development of EGT in horse wound repair.

Human chorionic gonadotropin-dependent induction of an equine aldo-keto reductase (AKR1C23) with 20alpha-hydroxysteroid dehydrogenase activity during follicular luteinization in vivo.

Aldo-keto reductases (AKRs) are multifunctional enzymes capable of acting on a wide variety of substrates, including sex steroids. AKRs having 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD) activity can reduce progesterone to 20alpha-hydroxy-4-pregnen-3-one (20alpha-DHP), a metabolite with lower affinity for the progesterone receptor. The objective of this study was to investigate the regulation of equine AKR1C23 during human chorionic gonadotropin (hCG)-induced ovulation/luteinization. The equine AKR1C23 cDNA was cloned and shown to encode a 322 amino acid protein that is conserved (71-81% identity) when compared with mammalian orthologs. RT-PCR/Southern blotting analyses were performed to study the regulation of AKR1C23 transcripts in equine preovulatory follicles isolated between 0 and 39 h after hCG treatment (ovulation occurring 39-42 h post-hCG). Results showed the presence of low AKR1C23 expression before hCG treatment, but a marked increase was observed in follicles obtained 12 h after hCG (P<0.05). Analyses of isolated preparations of granulosa and theca interna cells identified low mRNA expression in both cell types prior to hCG treatment, with granulosa cells clearly being the predominant site of follicular AKR1C23 mRNA induction. A specific polyclonal antibody was raised against a fragment of the equine protein and immunoblotting analyses showed an increase in AKR1C23 protein in granulosa cell extracts when comparing follicles isolated at 36 h post-hCG vs those collected prior to treatment, in keeping with mRNA results. Immunohistochemical data confirmed the induction of the enzyme in follicular cells after hCG treatment. The enzyme was tested for 20alpha-HSD activity and was shown to exhibit a K(M) of 3.12 microM, and a V(max) of 0.86 pmol/min per 10 microg protein towards progesterone. The levels of 20alpha-DHP measured in follicular fluid reflected this activity. Collectively, these results demonstrate for the first time that the gonadotropin-dependent induction of follicular luteinization is accompanied by an increase in AKR1C23 expression. Considering the 20alpha-HSD activity of AKR1C23, its regulated expression in luteinizing preovulatory follicles may provide a biochemical basis for the increase in ovarian 20alpha-DHP observed during gonadotropin-induced luteinization/ovulation. (The nucleotide sequence reported in this paper has been submitted to GenBank with accession number AY955082.).

Size-selection of cDNA libraries for the cloning of cDNAs after suppression subtractive hybridization.

Here we describe the establishment of size-selected cDNA libraries for the cloning of full-length cDNAs that were initially identified by suppression subtractive hybridization (SSH) technology as being differentially expressed. First, the SSH-cDNA fragments were used as 32P-probes to verify their level and differential pattern of expression by virtual Northern and to establish their corresponding full-length cDNA size. Second, cDNAs were separated by size on agarose gels and used to construct size-selected cDNA plasmid libraries, which were then screened by colony hybridization with the SSH-cDNA fragments. We conclude that the described approach complements SSH technology by allowing efficient cloning and characterization of the corresponding full-length cDNA from any desired cell type or species. This approach will give researchers the ability to specifically target and study differentially expressed genes in an efficient manner for functional genomic studies.

Presence of LH receptor mRNA in granulosa cells as a potential marker of oocyte developmental competence and characterization of the bovine splicing isoforms.

As the expression of the LH receptor (LH-R) in granulosa cells is thought to be associated with later stages of folliculogenesis, this study was undertaken to evaluate the presence of LH-R mRNA as a suitable marker for developmental competence of oocytes. Granulosa cells and cumulus-oocyte complexes (COCs) were recovered from cows that had received ovarian stimulation. The COCs were subjected to embryo production procedures in vitro to assess the embryonic potential of the oocyte, and the corresponding granulosa cells were used to evaluate the presence of LH-R mRNA by RT-PCR. The presence of LH-R transcripts in granulosa cells is not a key characteristic of a follicle bearing a competent oocyte, although a higher proportion of oocytes reach the blastocyst stage when LH-R mRNA is detected in the granulosa cells. Different LH-R isoforms were cloned and sequence discrepancies among six of the isoforms enabled the design of specific oligonucleotides to study the presence of the isoforms in different follicular cells. All LH-R transcripts studied and the 80 kDa protein product corresponding to the full length receptor were found in granulosa cells of small (< 4 mm) and large (> 5 mm) follicles. When the granulosa cells were cultured, the transcripts were downregulated by the culture conditions; downregulation was more acute in granulosa cells from small follicles. The addition of LH to the culture media enhanced LH-R mRNA downregulation. The presence of several LH-R transcript isoforms was tissue specific and in the theca cells LH-R mRNA was restricted mainly to cells from larger follicles. This finding indicates that the expression and the splicing of LH-R mRNA are regulated in a cell-specific and follicular size-specific manner.

Molecular cloning and induction of bovine prostaglandin E synthase by gonadotropins in ovarian follicles prior to ovulation in vivo.

Prostaglandin E(2) (PGE(2)) is thought to be an ultimate prostaglandin effector during the ovulatory process, and the objectives of this study were to clone bovine PGE synthase (PGES) and to characterize its regulation by gonadotropins in preovulatory follicles in vivo. The bovine PGES complementary DNA (cDNA) was shown to contain a 5'-untranslated region of eight base pairs (bp), an open reading frame of 462 bp and a 3'-untranslated region of 406 bp. The putative bovine PGES open reading frame encodes a 153-amino acid protein that is 85, 78, and 78% identical to the human, rat, and mouse PGES homologs, respectively. The regulation of PGES during ovulation was studied using three different models in vivo: 1) human chorionic gonadotropin (hCG)-induced ovulation during a normal estrous cycle; 2) hCG-induced ovulation following ovarian hyperstimulation; and 3) spontaneous ovulation during natural estrus. Results from semi-quantitative reverse transcription-polymerase chain reaction/Southern blotting analyses showed that the hCG/luteinizing hormone surge caused a significant increase in PGES mRNA. Levels of PGES transcripts were low or undetectable prior to hCG/luteinizing hormone but increased markedly 18-24 h after hCG in models 1 and 2, and 18-24 h after the onset of natural estrus in model 3 (p < 0.05). Analyses on isolated preparations of granulosa and theca interna cells indicated that the granulosa cell layer was the predominant site of follicular PGES expression. The regulation of the protein was studied in the same models using a specific antibody raised against a fragment of bovine protein (Delta PGES; from Glu(49) to Val(146)). Results from immunoblots showed an induction of bovine PGES (M(r) = 17,000) 18-24 h after hCG treatment or onset of estrus (p < 0.05). The protein was detected in extracts of granulosa cells but not in theca interna. Collectively, these results demonstrate that the ovulatory process is associated with a gonadotropin-dependent induction of PGES in granulosa cells of ovarian follicles in vivo, thus establishing for the first time the regulation of the enzyme in a physiological context.

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.

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.

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.

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.

Levels of messenger RNA encoding ovarian receptors for FSH and LH in cattle during superovulation with equine chorionic gonadotrophin versus FSH.

This study tested the hypothesis that luteal LH receptor (LHr) and follicular LHr and FSH receptor (FSHr) steady-state mRNA levels are greater during superovulation with equine chorionic gonadotrophin (eCG) compared with that with FSH. Heifers were stimulated with eCG (n = 10) or FSH (n = 10), and ovaries were recovered the day before and at 12 and 24 h after luteolysis was induced with prostaglandin F2 alpha (PGF2 alpha). Total RNA was purified from individual follicles and corpora lutea. Steady-state levels of LHr and FSHr mRNA were assessed by slot blot analysis employing homologous cDNA probes. There were no differences in luteal LHr between FSH- and eCG-stimulated animals before luteolysis, and hybridization signals were detected in only one of six animals by 12 h after injection of PGF2 alpha. After PGF2 alpha injection, steady-state levels of follicular LHr were 4-fold lower (P < 0.05) and follicular FSHr mRNA levels were 2.4-fold lower (P < 0.05) in eCG- compared with FSH-treated cattle. In eCG-treated animals, induction of luteolysis led to a significant increase in follicular LHr mRNA levels (P < 0.01) and a significant decrease in follicular FSHr mRNA levels (P < 0.01). There was no such effect of luteolysis in FSH-treated animals. We conclude that superovulation with eCG, compared with FSH, results in lower follicular levels of LHr and FSHr mRNA but does not affect luteal LHr mRNA levels.

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.

Changes in morphological appearance and functional capacity of recruited follicles in cows treated with FSH in the presence or absence of a dominant follicle.

This study was designed to determine the effect of the presence of a dominant follicle at the beginning of FSH stimulation on the morphological appearance and functional capacity of recruited follicles during FSH stimulation in cattle. Synchronized nonlactating dairy cows were assigned to 1 of 2 groups and treated with FSH in the presence (n = 5) or absence (n = 6) of a dominant follicle between Days 7 and 12 of the estrous cycle (Day 0 = estrus) to stimulate follicular growth. Dominant follicles were identified by daily ultrasonographic observations, beginning on Day 3 of the estrous cycle. Dominant follicle had an ultrasonographic diameter > or = 10 mm and were in a growing phase, or maintaining a constant diameter (> or = 10 mm) for less than 4 d. Ovaries were collected at slaughter on the morning of the third day following initiation of the FSH stimulation. All follicles > 2 mm were dissected, classified according to diameter (Class 1: 2 to 4.4 mm; Class 2: 4.5 to 7.9 mm; Class 3: > 8 mm), and incubated individually for 90 min in medium M-199 (37 degrees C, 5% CO2). Following incubation, integrity of each follicle was evaluated histologically to assess the level of atresia and biochemically to determine the in vitro release of estradiol (E2) and androstenedione in culture media. On Day 3 of the FSH treatment, mean number of follicles in each class was similar (P > 0.1) between the 2 groups. The percentage of atretic follicles in Classes 1 and 3 on Day 3 of the FSH stimulation did not differ (P > 0.1) between the 2 groups. However, the percentage of atretic follicles in Class 2 was higher (P < 0.005) in cows treated with FSH in presence than in absence of a dominant follicle (60.8 vs 38.2%). The release of E2 in culture media by small Class 1 atretic or healthy follicles, by Class 2 atretic and by Class 3 healthy follicles was not affected (P > 0.1) by the ovarian status. However (P < 0.001), the release of E2 in culture media of Class 2 healthy and Class 3 atretic follicles was less for follicles harvested from cows bearing than from those not bearing a dominant follicle. Within each follicular class, concentrations of androstenedione in the culture media did not differ between the 2 groups (P > 0.1). These results suggest that the presence of a dominant follicle at the beginning of FSH stimulation alters the population of follicles recruited FSH stimulation. This may be associated with the reported decrease of the superovulatory response in cows superovulated in presence of a dominant follicle.

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.

Follicular development and reproductive endocrinology during and after superovulation in heifers and mature cows displaying contrasting superovulatory responses.

To understand the causes for poor response to superovulation in mature cows of high genetic potential, endocrine and follicular events during and after superovulation were compared in heifers (<2 yr old) yielding large numbers of embryos and cows (9 to 13 yr old) known to be poor embryo donors. Follicular development was monitored by daily ultrasonography. Blood samples were taken 2 to 3 times a day for the measurements of P4, E2, FSH and LH by RIA. Intensive blood collections at 15-min intervals for 6 h were also performed during preovulatory and luteal phases. The number of embryos produced in the heifers (15.2 +/- 2; mean +/- SEM) and the cows (0.6 +/- 0.4), was similar to the number of ovulatory follicles derived from ultrasonographic observations in the heifers (16.2 +/- 3.7), but not in the cows (7.8 +/- 2.8). Contrary to that observations in heifers, there was no increase in the number of 4- to 5-mm follicles in cows during superovulation. The number of larger follicles (>5 mm) increased during superovulation in both cattle groups, but it was significantly lower in cows than in heifers. During superovulation, the maximal E2 concentration was greater (P < 0.0001) in heifers than in cows. One cow showed delayed luteolysis during superovulation, while another had abnormally high FSH (>10 ng/ml) and LH (>3 ng/ml) concentrations following superovulation. All the cows had a postovulatory FSH rise which was not detected in the heifers. The results showed that attempts to improve superovulatory response in mature genetically valuable cows are hampered by a number of reproductive disorders that are not predictable from the study of the unstimulated cycle.

Follicular development and reproductive endocrinology during a synchronized estrous cycle in heifers and mature cows displaying contrasting superovulatory responses.

Ovarian follicular development and plasma concentrations of progesterone (P4), estradiol-17 beta (E2), luteinizing hormone (LH), and follicle-stimulating hormone (FSH) were compared during a synchronized estrous cycle between heifers and mature cows displaying contrasting superovulatory responses. Six heifers < 2 years old with a history of good responses to superovulatory (SOV) treatment and six cows 9 to 13 years old with poor responses to SOV treatments were used. Follicular development was monitored by daily ultrasonography. Blood samples were collected two to three times daily for P4 and E2 and thrice daily for LH and FSH analysis. Intensive sampling (samples every 15 min for 6 hr) was performed at critical periods of follicular development to analyze the pulsatile secretion of gonadotropins. In both cattle groups, a transient increase (P = 0.0001) in E2 occurred 4 to 5.7 d after the preovulatory LH surge or 2.3 d before the dominant follicle reached its maximum size. FSH concentrations increased (P = 0.006) before the emergence of the second cohort of follicles and then decreased despite no change in the concentration of E2. Contrary to our expectation and despite differences between groups in terms of age, number of previous SOV treatments, and divergent responses to superovulation, follicular development was similar in both groups. However, during the luteal phase, concentrations of E2 and FSH and LH pulse amplitudes were less (P < or = 0.05) in cows than in heifers. Therefore, follicular development monitored by ultrasonography and endocrine profiles during a synchronized estrous cycle are of limited value to predict quality of embryo donors.

Bovine SRY gene locus: cloning and testicular expression.

The bovine SRY gene was cloned by a combination of anchored polymerase chain reaction (PCR) amplification of genomic restriction fragments and reverse transcription-PCR (RT-PCR) of testicular RNA. We report 1800 bp of combined genomic and cDNA sequences including 911 bp of 5' upstream sequences, an open reading frame of 687 bp, and 202 bp of sequences corresponding to the 3' end of the mRNA. The bovine SRY gene encodes a deduced (predicted on the basis of a cDNA sequence) protein product of 229 amino acids, with sequence conservation between species, notably in the region of the high-mobility group (HMG) domain or HMG box. Outside of the HMG box, the bovine SRY structure shows greater resemblance to the human SRY than to the mouse Sry. As with human SRY promoter sequences, putative binding sites for Sp1 and for SRY itself are seen in the bovine SRY promoter region. Unlike the human SRY promoter, CAAT and TATA box motifs are present in the bovine sequences. Southern analysis and PCR amplification of male and female bovine genomic DNA show that the described sequences are specific to the Y chromosome. Northern analysis of bull testicular RNA demonstrated low levels of expression of the bovine SRY gene in adult testes with a major poly(A) species at 1.9 kb. RT-PCR amplification of bull testicular RNA revealed multiple sites of polyadenylation, but sequencing showed no consensus polyadenylation signal.

Bovine activin receptor type II cDNA: cloning and tissue expression.

The cDNA encoding the bovine activin type II receptor has been cloned by reverse transcription-polymerase chain reaction (RT-PCR) amplification of a bovine testicular RNA preparation. Sequence comparisons of the bovine activin type II receptor with its human, mouse and rat homologues show strong evolutionary conservation at the nucleotide level of 94.9%, 93.5%, 92.9% and at the amino acid level of 98.6%, 99.0%, 98.8%, respectively. Bovine activin type II receptor mRNA is widely but not strongly expressed in reproductive tissues, with a major RNA band at 6 kb and minor bands at 5 kb and 3 kb. The differential levels of expression observed in these tissues suggest that levels of bActRII gene expression are regulated. Furthermore, we have observed decreasing levels of the bovine activin type II receptor mRNA with testes maturation.