Identification and characterization of a nuclear factor-κ B-p65 proteolytic fragment in nuclei of porcine hepatocytes in monolayer culture.

Hepatic responses to proinflammatory signals are controlled by the activation of several transcription factors, including, nuclear factor-κ B (NF-κB). In this study, hepatocytes prepared from suckling pigs and maintained in serum-free monolayer culture were used to define a novel proinflammatory cytokine-specific NF-κB subunit modification. The immunoreactive p65 protein was detected by Western blot analysis at the appropriate molecular weight in the cytosol of control cultures and those incubated with tumor necrosis factor-α (TNF). However, in nuclei, the p65 antisera cross-reacted with a protein of approximately 38 kDa (termed p38) after TNF addition, which was not observed in the cytosol of control or cytokine-treated cells. Specifically, incubation with TNF also resulted in phosphorylation (P < 0.05) of the inhibitor complex protein (IκB), whereas incubation with other cytokines, IL-6, IL-17a, or oncostatin M was not associated with either phosphorylation of IκB or nuclear translocation of p65. Intracellular endothelial nitric oxide synthase was deceased (P < 0.05) and plasminogen activator inhibitor-1 secretion was increased (P < 0.05) after TNF incubation. The TNF-induced p38 protein was purified from hepatocyte nuclei by immunoprecipitation, concentrated by electrophoresis, and subsequently analyzed by mass spectrometry. Ten unique NF-κB p65 peptides were identified after digestion with trypsin and chymotrypsin; however, all were mapped to the N-terminus and within the first 310 amino acid residues of the intact p65 protein. Although low molecular weight immunoreactive p65 molecules were previously observed in various human and rodent systems, this is the first report to positively identify the p38 fragment within hepatocyte nuclei or after specific cytokine (TNF) induction.

Growth and Development Symposium: Development, characterization, and use of a porcine epiblast-derived liver stem cell line: ARS-PICM-19.

Totipotent embryonic stem cell lines have not been established from ungulates; however, we have developed a somatic stem cell line from the in vitro culture of pig epiblast cells. The cell line, ARS-PICM-19, was isolated via colony cloning and was found to spontaneously differentiate into hepatic parenchymal epithelial cell types, namely hepatocytes and bile duct cells. Hepatocytes form as monolayers and bile duct cells as 3-dimensional bile ductules. Transmission electron microscopy revealed that the ductules were composed of radially arranged, monociliated cells with their cilia projecting into the lumen of the ductule whereas hepatocytes were arranged in monolayers with lateral canalicular structures containing numerous microvilli and connected by tight junctions and desmosomes. Extensive Golgi and rough endoplasmic reticulum networks were also present, indicative of active protein synthesis. Analysis of conditioned medium by 2-dimensional electrophoresis and mass spectrometry indicated a spectrum of serum-protein secretion by the hepatocytes. The PICM-19 cell line maintains a range of inducible cytochrome P450 activities and, most notably, is the only nontransformed cell line that synthesizes urea in response to ammonia challenge. The PICM-19 cell line has been used for several biomedical- and agricultural-related purposes, such as the in vitro replication of hepatitis E virus, a zoonotic virus of pigs, and a spaceflight experiment to evaluate somatic stem cell differentiation and liver cell function in microgravity. The cell line was also evaluated as a platform for toxicity testing and has been used in a commercial artificial liver rescue device bioreactor. A PICM-19 subclone, PICM-19H, which only differentiates into hepatocytes, was isolated and methods are currently under development to grow PICM-19 cells without feeder cells. Feeder-cell-independent growth will facilitate the study of mesenchymal-parenchymal interactions that influence the divergent differentiation of the PICM-19 cells, enhance our ability to genetically modify the cells, and provide a better model system to investigate porcine hepatic metabolism.

Iron dextran treatment does not induce serum protein carbonyls in the newborn pig.

Oxidation of serum proteins can lead to carbonyl formation that alters their function and is often associated with stress-related diseases. As it is recommended that all pigs reared in modern production facilities be given supplemental iron at birth to prevent anemia, and metals can catalyze the carbonylation of proteins, the primary objective of this study was to determine whether standard iron dextran treatment was associated with enhanced serum protein oxidation in newborn piglets. Piglets were treated with 100 mg of iron dextran intramuscularly either on the day of birth, or on the third day after birth. Blood samples were collected from piglets 48 or 96 h after treatment and serum was harvested. For quantification, serum protein carbonyls were converted to hydrazones with dinitrophenyl hydrazine and analyzed spectrophotometrically. To identify and determine relative distribution of carbonylated proteins, serum protein carbonyls were derivatized with biotin hydrazide, separated by two-dimensional polyacrylamide gel electrophoresis, stained with avidin-fluorescein and identified by mass spectrometry. The standard iron dextran treatment was associated with no increase in total oxidized proteins if given either on the first or third day of life. In addition, with a few noted exceptions, the overall distribution and identification of oxidized proteins were similar between control and iron dextran-treated pigs. These results indicate that while iron dextran treatment is associated with a marked increase in circulating iron, it does not appear to specifically induce the oxidation of serum proteins.

A calcium-activated nucleotidase secreted from Ostertagia ostertagi 4th-stage larvae is a member of the novel salivary apyrases present in blood-feeding arthropods.

Apyrases (ATP-diphosphohydrolase) comprise a ubiquitous class of glycosylated nucleotidases that hydrolyse extracellular ATP and ADP to orthophosphate and AMP. One class of newly-described, Ca2+-dependent, salivary apyrases known to counteract blood-clotting, has been identified in haematophagous arthropods. Herein, we have identified a gene (Oos-apy-1) encoding a protein that structurally conforms to the Ca2+-activated apyrase from the bed bug, Cimex lectularius, by immunologically screening an Ostertagia L4 cDNA expression library. The expressed protein (rOos-APY-1) was biochemically functional in the presence of Ca2+ only, with greatest activity on ATP, ADP, UTP and UDP. Host antibodies to the fusion protein appeared as early as 14 days post-infection (p.i.) and increased through 30 days p.i. Immunohistochemical and Western blot analyses demonstrated that the native Oos-APY-1 protein is present in the glandular bulb of the oesophagus and is confined to the L4. A putative signal sequence at the N-terminus and near 100% identity with a Teladorsagia circumcincta L4 secreted protein is consistent with the native protein being secreted at the cellular level. Predicated upon substrate specificity, the native protein may be used by the parasite to control the levels of host extracellular nucleotides released by locally-damaged tissues in an effort to modulate immune intervention and inflammation.

Proteomic analyses of male contributions to honey bee sperm storage and mating.

Honey bee (Apis mellifera L.) queens mate early in life and store sperm for years. Male bees likely contribute significantly to sperm survival. Proteins were extracted from seminal vesicles and semen of mature drones, separated by electrophoresis, and analysed by peptide mass fingerprinting. Computer searches against three databases, general species, honey bees and fruit flies, were performed. Spectra were used to query the recently generated honey bee genome protein list as well as general species and fruit fly databases. Of the 69 unique honey bee proteins found, 66 are also in Drosophila melanogaster. Two proteins only matched honey bee genes and one is a widespread protein lost from the fly genome. There is over-representation of genes implicated in the glycolysis pathway. Metabolism-associated proteins were found primarily in the seminal vesicle. Male accessory gland proteins as identified in Drosophila rarely had orthologs among proteins found in the honey bee. A complete listing of gel spots chosen including honey bee genome matches and Mascot searches of MALDI-TOF results with statistics is in the Supplementary table. MALDI-TOF spectra and more complete Mascot peptide mass fingerprinting data are available on request. Supplementary figs 1-3 show the stained protein gels.

Hormonal regulation of leptin receptor expression in primary cultures of porcine hepatocytes.

A study was conducted to elucidate hormonal control of leptin receptor gene expression in primary cultures of porcine hepatocytes. Hepatocytes were isolated from pigs (52 kg) and seeded into collagen-coated T-25 flasks. Monolayer cultures were established in medium containing fetal bovine serum for 1 day and switched to a serum-free medium for the remainder of the 3-day culture period. To establish basal conditions hepatocytes were maintained in serum-free William's E medium containing 10 nM dexamethasone and 1 ng/ml insulin. For the final 24 h, insulin (1 or 100 ng/ml) or glucagon (100 ng/ml), were added in the presence or absence of 100 nM triiodothyronine (T3). RNA was extracted and quantitative RT-PCR was performed with primers specific for the long form and total porcine leptin receptors. Leptin receptor expression was calculated relative to co-amplified 18S rRNA. Expression of the long form of the leptin receptor was confirmed under basal conditions. Insulin, glucagon and synthetic human proteins (ghrelin and GLP-1) at 100 ng/ml had no influence on leptin receptor expression; the addition of T3 was associated with a marked increase (P < 0.001) in expression of total and long forms of the leptin receptor by 1.6 and 2.4-fold, respectively. Addition of leptin to cells which were pre-treated with T3 for 24 h (to up-regulate leptin receptor expression), confirmed the lack of a direct effect of leptin on glucagon-induced glycogen turnover and cAMP production. These data suggest that porcine hepatocytes may be insensitive to leptin stimulation even when leptin receptor expression is enhanced by T3.

Immunohistochemical localization of chromogranin A in gonadotrophs and somatotrophs of the turkey and chicken pituitary.

In the course of producing monoclonal antibodies to turkey prolactin, three monoclonal antibodies to turkey chromogranin A (CgA) were also produced, apparently arising from minor contamination of the turkey prolactin immunogen with peptide fragments of CgA. The identity of the antigen recognized by these antibodies was established by tandem mass spectrometry de novo sequencing of seven tryptic peptides from a turkey pituitary protein purified by immunoaffinity chromatography. These peptides showed high homology with distinctly separate regions of mammalian and ostrich CgA, and in silico cloned chicken CgA sequences. Chromogranin A immunostaining patterns on Western blots and pituitary tissue sections differed from those of prolactin, growth hormone, or luteinizing hormone (LH). Dual-label fluorescent immunohistochemistry revealed that CgA was co-localized with LH in most avian gonadotrophs in young chickens and turkeys, but not in adult, laying birds. Conversely, CgA was found in a majority of somatotrophs in laying birds but was absent from somatotrophs in young, growing chickens and turkeys. Lactotrophs contained no detectable CgA immunoreactivity in the tissues studied. These results suggest that CgA may modulate hormone secretion by gonadotrophs and somatotrophs in a manner that differs between cell type with age or reproductive state.

Segregation of spermatozoa within sperm storage tubules of fowl and turkey hens.

In avian species, spermatozoa reside in the oviduct for prolonged periods in specialized structures known as sperm storage tubules, but little is known about the relative distribution of spermatozoa in these tubules after successive inseminations by different males. The staining efficacies of various fluorescent dyes for fowl and turkey spermatozoa were evaluated to investigate one proposed mechanism of sperm competition. Hens were then inseminated at different intervals with stained and unstained spermatozoa to observe the spatial distribution of spermatozoa within the storage tubules. Several novel fluorescent lipophilic tracers that successfully stain mammalian spermatozoa either did not stain fowl or turkey spermatozoa, or greatly impaired sperm motility. In contrast, Hoechst 33342 readily stained sperm nuclei (fowl: 25 nmol l-1; turkey: 77 nmol l-1) within 4 h without inhibiting sperm motility, or affecting fertility or the hatching ability of the eggs. Hens were tandemly inseminated with equal numbers of stained or unstained spermatozoa at 24 h intervals and were killed 24 h after the final insemination to study sperm entry and storage within the tubules. Oviductal mucosa containing sperm storage tubules was removed, and individual tubules were classified as containing stained spermatozoa, unstained spermatozoa, a mixture of stained and unstained spermatozoa, or as not containing spermatozoa. Results from the present study indicate that spermatozoa from two different inseminations generally segregate into different storage tubules in both fowl and turkey hens. Storage tubules containing mixed populations of spermatozoa were found in only 4% of fowl and 12% of turkey storage tubules examined. Thus, the mechanism of last-male precedence does not appear to be due to the stratification of spermatozoa within the tubules.

Flow cytometric sperm sorting: effects of varying laser power on embryo development in swine.

This study was conducted to determine fertilization rate and embryo development using the Beltsville Sperm Sexing Technology with two different laser power outputs, 25 and 125 milliwatts (mW). Freshly ejaculated boar semen was diluted; one aliquot was not stained or sorted (nonsort) and a second aliquot was stained with Hoechst 33342 and sorted as a complete population, not separated into X and Y populations (all-sort). Ovulation controlled gilts were surgically inseminated with 2 x 10(5) spermatozoa (44-46 hr after human chorionic gonadotropin (hCG)) into the isthmus of each oviduct, one oviduct receiving nonsort and the other all-sort at 25 or 125 mW. A total of 426 embryos were flushed from oviducts at slaughter 43 hr after laparotomy and prepared for determination of fertilization and cleavage rates using confocal laser microscopy for analysis of actin cytoskeleton and chromatin configuration. The percentage of fertilized eggs and embryos was less for the 25 mW all-sort compared to nonsort or the 125 mW all-sort (77.9 vs. 96.3 and 96.2%, P < 0.05). The percentage of fragmented embryos was greater for the 25 mW all-sort than the nonsort (15.2 vs. 4.5%, P < 0.05), but did not differ significantly from 125 mW all-sort mean (7.2%). The percentage of normal embryos (80.4% overall) did not differ (P > 0.05) among treatments. However, the rate of embryo development was slower (P < 0.05) after insemination with the 25 mW all-sort spermatozoa compared to nonsort spermatozoa. Embryos in the 3-4 and 5-9 cell stages for the 25-mW all-sort and nonsort were 78 and 20% vs. 49 and 50%, respectively. The embryo percentages for the 125 mW (3-4 and 5-9 cell stages, 59 and 35%) did not differ significantly (P > 0.05) from the nonsort or 25 mW all-sort. We conclude that the use of 125 mW laser power for sorting boar spermatozoa is advantageous to maintain high resolution separation and has no detrimental effect on embryo development compared to 25 mW.

Ultrastructure of the embryonic stem cells of the 8-day pig blastocyst before and after in vitro manipulation: development of junctional apparatus and the lethal effects of PBS mediated cell-cell dissociation.

Ultrastructural examination of 8-day hatched pig blastocysts (large and small), their cultured inner cell mass (ICM), and cultured epiblast tissue (embryonic stem cells) was undertaken to assess the development of epiblast cell junctions and cytoskeletal elements. In small blastocysts, epiblast cells had no desmosomes or tight junction (TJ) connections and few organized microfilament bundles, whereas in large blastocysts the epiblast cells were connected by TJ and desmosomes with associated microfilaments. ICM isolation by immunodissection damaged the endoderm cells beneath the trophectoderm cells but did not appear to damage the epiblast cells or their associated endoderm cells. Epiblast cells in cultured ICMs were similar in character to those in the intact large blastocyst except that perinuclear microfilaments were observed. Isolated pig epiblasts, cultured for approximately 36 hr on STO feeder layers, formed a monolayer whose cells were connected by TJ, adherens junctions and desmosomes with prominent microfilament bundles running parallel to the apical cytoplasmic membranes. Perinuclear microfilaments were a consistent feature in the approximately 36 hr cultured epiblast cells. A feature characteristic of differentiation into notochordal cells, i.e., a solitary cilium, was also observed in the cultured epiblast. Exposure of the cultured epiblast cells to Ca(++)-Mg(++)-free phosphate buffered saline (PBS) for 5-10 min resulted in extensive cell blebbing and lysis. The results may indicate that pig epiblast cells could be more easily dissociated from early blastocysts ( approximately 400 microm in diameter) if immunodissection damage to the ICM can be avoided. It may be difficult, however, to establish them as embryonic stem cell lines because the cultured pig epiblast cells were easily lysed by standard cell-cell dissociation methods.

Apoptosis during folliculogenesis in pigs.

The number of female germ cells in pig fetuses decreases by 70% between day 50 after mating and day 300 after birth. Approximately 55% of antral follicles undergo degeneration (atresia) except during the 3 days before oestrus, when only 15% of the follicles survive to ovulate. Apoptosis, a form of programmed cell death, is recognized as the mechanism of germ cell death and follicle atresia at all stages of folliculogenesis. The internucleosomal cleavage of genomic DNA caused by caspase-induced deoxyribonuclease activity was measured in pig granulosa cells by DNA fluorescence flow cytometry, densitometry of fluorescently labelled internucleosomal DNA fragments and immunohistochemical analysis of the 3' end labelling of deoxyribonuclease-nicked DNA on frozen tissue sections. Follicular atresia during the 3 days before oestrus is associated with a 60-70% decrease in the secretion of FSH. In granulosa cells, apoptosis is associated with decreased cell proliferation and reduced production of oestradiol and inhibin. In cultured pig granulosa cells, FSH and IGF-I are anti-apoptotic and a caspase inhibitor blocked apoptosis, thereby providing evidence of caspase activity. Oocytes in most follicles have resumed meiotic maturation; therefore, one role for apoptosis and follicle atresia may be to act as a barrier to ovulation of oocytes that have not remained in meiotic arrest.

Inhibition of apoptosis in cultured porcine granulosa cells by inhibitors of caspase and serine protease activity.

Protease inhibitors were used to test the hypothesis that caspases and other proteases were active during apoptosis in cultured porcine granulosa cells. Cells isolated from 3 to 6 mm follicles were cultured for 24 h in Dulbecco's modified Eagles medium: Hams F12 (1:11 containing 1% fetal bovine serum. Final inhibitor concentrations, added in 10 microL of dimethylsulfoxide, were 0, 1, 5, 25 and 125 microM. Cells with compromised plasma membrane integrity, identified by uptake ethidium homodimer, increased during culture in the absence of inhibitors from 37% to 43%. Apoptotic (A0) cells, identified by DNA fluorescence flow cytometry, increased (P < 0.05) from 1.7% to 29%. The serine protease inhibitor N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) at 125 microM was lethal increasing (P < 0.05) cells with compromised membranes to 92%. In response to TPCK, A0 cells decreased from 55% to 1.2%; progesterone and estradiol production were decreased by 94% and 98%, respectively. The general caspase inhibitor, benzyloxycarbonyl-valinyl-alaninyl-aspartyl fluoro methylketone, decreased (P < 0.05) A0 cells linearly from 33% to 3 % between 0 and 125 microM without significant effect on steroidogenesis or on the percentage of cells with compromised plasma membranes. Other inhibitors only had a marginal effect on apoptosis; concentrations of > or = 1 microM decreased (P < 0.05) A0 cells from 29% to 18% to 21% and had no significant effect on membrane integrity or steroid production. We conclude that caspases are associated with apoptosis in cultured porcine granulosa cells. Death induced by TPCK was through a non-apoptotic mechanism.

Changes in porcine oocyte germinal vesicle development as follicles approach preovulatory maturity.

This study was conducted to determine the distribution of oocytes in meiotic arrest as a function of follicle maturation, atresia status, and follicular fluid steroid concentrations. Oocytes (n = 138) from > or = 3 mm follicles were recovered from gilts (n = 3/d) on Days 1, 3, 5, and 7 of the follicular phase initiated by withdrawal of altrenogest treatment. They were fixed in 4% paraformaldehyde, stained with Hoechst 33342, and examined by laser scanning confocal microscopy using combined bright field Nomarski optics and ultraviolet laser illumination. The number of oocytes in complete meiotic arrest increased (P < 0.05) as a function of the stage of maturation from 29% on Day 1 to 79 and 67% on Days 3 and 5, respectively. Oocytes showing complete germinal vesicle breakdown (GVBD) were found only on Day 7 (24 to 36 h after the preovulatory LH surge). The distribution of GV stages on Days 1 to 5 did not differ between atretic (n = 27) and nonatretic follicles (n = 81). In nonatretic follicles, GV stage was inversely related to the concentration of estradiol on Day 7 and to the concentrations of progesterone and androstenedione (P < 0.05) on Days 5 and 7 indicating that meiotically arrested oocytes were likely to be found in follicles with highest levels of steroidogenesis. In conclusion, a large proportion of oocytes present in 3 to 5 mm follicles had begun GVBD. The follicles in the ovulatory cohort may be recruited or selected from preexisting 3 to 5 mm follicles, or younger population with oocytes that are in complete meiotic arrest.

Comparative map alignment of BTA27 and HSA4 and 8 to identify conserved segments of genome containing fat deposition QTL.

Quantitative trait loci (QTL) associated with fat deposition have been identified on bovine Chromosome 27 (BTA27) in two different cattle populations. To generate more informative markers for verification and refinement of these QTL-containing intervals, we initiated construction of a BTA27 comparative map. Fourteen genes were selected for mapping based on previously identified regions of conservation between the cattle and human genomes. Markers were developed from the bovine orthologs of genes found on human Chromosomes 1 (HSA1), 4, 8, and 14. Twelve genes were mapped on the bovine linkage map by using markers associated with single nucleotide polymorphisms or microsatellites. Seven of these genes were also anchored to the physical map by assignment of fluorescence in situ hybridization probes. The remaining two genes not associated with an identifiable polymorphism were assigned only to the physical map. In all, seven genes were mapped to BTA27. Map information generated from the other seven genes not syntenic with BTA27 refined the breakpoint locations of conserved segments between species and revealed three areas of disagreement with the previous comparative map. Consequently, portions of HSA1 and 14 are not conserved on BTA27, and a previously undefined conserved segment corresponding to HSA8p22 was identified near the pericentromeric region of BTA8. These results show that BTA27 contains two conserved segments corresponding to HSA8p, which are separated by a segment corresponding to HSA4q. Comparative map alignment strongly suggests the conserved segment orthologous to HSA8p21-q11 contains QTL for fat deposition in cattle.

In situ analysis of the changes in expression of ovarian inhibin subunit mRNAs during follicle recruitment after ovulation in pigs.

In situ hybridization was used on frozen tissue sections with digoxigenin-labelled antisense riboprobes to inhibin/activin alpha and beta(A) subunits to determine whether inhibin/activin subunit mRNA expression was associated with development of growing, steroidogenically active follicles during follicle recruitment after ovulation. Cell proliferation-associated nuclear antigen Ki-67 protein and cytochrome P450 aromatase expression in granulosa cells were determined immunohistochemically and used as markers for granulosa cell proliferation and steroidogenesis, respectively, on days 3, 5 and 7 after the onset of oestrus. The amounts of inhibin/activin alpha and beta(A) subunit mRNA and P450 aromatase protein were greater (102, 93, and 238%, respectively; P < 0.05) in medium than in small non-atretic follicles and were positively correlated with Ki-67 and with each other. Inhibin/activin alpha and beta(A) mRNA, P450 aromatase, and Ki-67 in granulosa cells were reduced by 66-83% (P < 0.001) in atretic follicles compared with non-atretic follicles. In addition, inhibin/activin alpha and beta(A) mRNA and P450 aromatase in small (1-2 mm) non-atretic follicles decreased (P < 0.05) between day 3 and day 7 independently of morphological or biochemical signs of atresia. The pattern of inhibin/activin subunit mRNA expression supports the notion that activin and inhibin have roles in growth and steroidogenesis in follicle recruitment during the early luteal phase of the oestrous cycle.

Absence of correlation between in situ expression of cytochrome P450 17alpha hydroxylase/lyase and 3beta-hydroxysteroid dehydrogenase/(Delta5-4) isomerase messenger ribonucleic acids and steroidogenesis during pubertal development in the rat testis.

Changes in expression of Leydig cell 3beta-hydroxysteroid dehydrogenase (3betaHSD) and 17alpha-hydroxylase/C17-20 lyase (P450(17alpha)) messenger RNA (mRNA) during pubertal development have not been well characterized in the rat. In the present study, expression of 3betaHSD and P450(17alpha) were determined in frozen sections of testes of immature (days 21 and 28), pubertal (days 45 and 60) and adult (day 90) rats by in situ hybridization using digoxigenin-labeled riboprobes and quantified densitometrically. Measures of steroidogenesis in this study, 3betaHSD and P450(17alpha) enzyme activities per testis and plasma testosterone concentration, increased during pubertal development, peaking at 45-60 days of age. Expression of 3betaHSD protein, a marker for Leydig cell function, was abundantly immunolocalized to the interstitial compartment of the testis. Quantified densitometrically, the amount of 3betaHSD protein did not vary significantly during pubertal development. Transcripts of 3betaHSD and P450(17alpha) were expressed abundantly by clusters of immature Leydig cells in immature animals. However, in contrast to measures of steroidogenesis during pubertal development, mRNA of 3betaHSD and P450(17alpha) decreased to undetectable levels at the age of 45 and 60 days, respectively. The decline in mRNA of 3betaHSD and P450(17alpha) was confirmed by Northern analysis. Expression of 3betaHSD and P450(17alpha) transcripts rebounded in the adult at 90 days and were comparable to levels of expression observed in immature animals. These results show that during pubertal development the steady-state accumulation of mRNA of 3betaHSD and P450(17alpha) are not correlated with accumulation of 3betaHSD protein, enzyme activities of 3betaHSD and P450(17alpha), or testosterone secretion. Possible explanations of the depletion of transcripts during pubertal development include: specific inhibition of transcription, increased mRNA instability, or high translational activity.

Localization and expression of low-density lipoprotein receptor, steroidogenic acute regulatory protein, cytochrome P450 side-chain cleavage and P450 17-alpha-hydroxylase/C17-20 lyase in developing swine follicles: in situ molecular hybridization and immunocytochemical studies.

The present study utilizes in situ molecular hybridization and immunocytochemistry to investigate the follicular localization and expression of four thematically related sterol-metabolizing genes; low-density lipoprotein (LDL) receptor, steroidogenic acute regulatory protein (StAR), cytochrome P450 side-chain cleavage (CYP11A) enzyme, and cytochrome P450 17alpha-hydroxylase/C(17-20) lyase (CYP17). To this end, semiquantitative analyses were applied to individual nonatretic follicles (N=54) harvested from cycling gilts slaughtered on days 1, 3, 5, and 7 (N=3 per day) following withdrawal of the progesterone agonist, altrenogest. In situ and immunocytochemical signal intensities were assigned numerical values of 0-3 corresponding to the degree of expression of each mRNA and its corresponding protein. LDL receptor mRNA and protein content was undectable in theca and granulosa cells on days 1, 3, and 5, and then increased to low levels in theca cells on day 7. StAR message rose progressively in theca cells with follicular maturation, reaching a maximum on day 5, and then declining slightly on day 7 after the LH surge. In granulosa cells, small amounts of StAR mRNA and protein were detected on days 5 and 7. The amounts of CYP11A mRNA and protein were high in theca cells, and increased at each time point studied. Granulosa cells exhibited minimal CYP11A message on days 3, 5, and 7, while protein became detectable at low levels on day 7 only. Expression of CYP17 was localized exclusively in theca cells with protein and message content increasing unidirectionally to maxima on days 5 (RNA) and 7 (protein), respectively. Follicular fluid concentrations of androstenedione, and progesterone in contralateral ovaries correlated strongly and positively with accumulation of CYP17, and CYP11A proteins. In summary, these analyses demonstrate that preovulatory follicular development proceeds with the coordinate induction of pivotal genes and proteins that mediate the selective uptake, delivery and utilization of sterol substrate in granulosa and theca-cell steroidogenesis.

Expression of bcl-2 and 3-beta hydroxysteroid dehydrogenase protein during oocyte and follicle development in fetal and post-natal pig ovaries.

The fetal and post-natal development of the pig ovary involves both proliferation and apoptotic loss of germ cells, follicle formation and growth, and the initiation of oocyte meiotic maturation. The present study measured the expression of the proto-oncogene Bcl-2 immunohistochemically on paraffin sections of pig ovaries to determine its relationship with folliculogenesis on Days 50 and 80 post coitum (p.c.) and on Days 1, 21, and 56 post partum (p.p.). The expression of the steroidogenic enzyme 3beta-hydroxysteroid dehydrogenase (3betaHSD) was used to determine the lineages of the cells forming the ovarian follicles, and the expression of the cell proliferation-associated nuclear antigen Ki-67 was used to determine germ cell proliferation and the initiation of follicle growth. Expression of Ki-67 showed that many oogonia were proliferating on Days 50 and 80 p.c. Granulosa cells were more proliferative on Day 56 p.p. than at any other stage; Ki-67 was expressed in 70% of growing follicles and granulosa cells had a 3% mean staining index per section. Less than 4% of germ cells and follicles had morphological signs of degeneration during the period of the study. Bcl-2 was most abundant on Days 21 p.p. and 56 p.p.; staining was localized to stromal cells among follicles and in small clusters in the cortical medullary junction (CMJ). 3BetaHSD staining on Day 50 p.c. was seen in cords of stromal cells within the medulla of the ovary, and in the stromal cells investing the oogonial nests. On Days 80 p.c., 1 p.p., 21 p.p., and 56 p.p., 3betaHSD was expressed in the granulosa cells of primary or primordial follicles at the CMJ. Production of Bcl-2 by somatic cells may support germ cell and preantral follicle survival.