The pregnancy-associated glycoproteins (PAG) constitute a large family of recently duplicated genes. They show structural resemblance to pepsin and related aspartic proteinases. A total of 21 bovine (bo) PAG and 9 ovine (ov) PAG cDNA have been identified. Phylogenetic analysis indicated that the PAG are divided into two main groupings that accurately reflect their tissue expression, as determined by in situ hybridization. In the first pattern, represented by ovPAG-2 and boPAG-2, -8, -10, and -11 (where the numbering is arbitrary and reflects order of discovery within species), expression occurred throughout the outer epithelial layer of the placenta (trophectoderm). The second pattern was predominant localization to binucleate cells. Ribonuclease protection assays, which allow discrimination between closely related transcripts, have shown that the expression of PAG varies in a temporal manner over pregnancy. Of those bovine PAG expressed predominantly in binucleate cells, boPAG-1, -6, and -7 are expressed weakly, if at all, by Day 25 placenta, but are present at the middle and end of pregnancy. Others, such as boPAG-4, -5, and -9, are expressed at Day 25 and at earlier stages. Although not among the earliest PAG produced by the trophoblast, boPAG-1 has been used for pregnancy diagnosis, particularly in dairy cows, where there is a major need for a sensitive method capable of detecting pregnancy within 1 mo of conception. It seems likely that some of the newly discovered PAG will be better candidates than PAG-1 for pregnancy diagnosis.

The objective of this study was to investigate changes in expression of mRNAs encoding FSH receptor (FSHr), LH receptor (LHr), cytochrome P450 side-chain cleavage (P450(scc)), cytochrome P450 17alpha-hydroxylase (P450(c17)), and cytochrome P450 aromatase (P450(arom)) during recruitment and selection of bovine ovarian follicles. Dairy heifers (4-5 per group) were ovariectomized at 12, 24, 36, 48, 60, 72, 84, or 96 h after initiation of the first follicular wave following estrus as determined by ultrasonography (Time 0 = initiation of follicular wave; mean +/- SEM = 42.0 +/- 2.6 h after estrus). Expression of mRNAs encoding FSHr, LHr, P450(scc), P450(c17), and P450(arom) was detected by in situ hybridization and quantified by image analysis. Antral follicles were classified as healthy or atretic. Healthy follicles expressed higher (p < 0.01) amounts of mRNAs for gonadotropin receptors and steroidogenic enzymes than did atretic follicles, and expression of LHr, FSHr, P450(scc), P450(c17), and P450(arom) increased (p < 0.01) with follicular size and stage of the follicular wave. Expression of mRNAs for P450(scc), P450(arom), and LHr was time- and size-dependent during recruitment and selection. During recruitment, expression of mRNAs for P450(scc) and P450(arom) was first detected in granulosa cells of 16 of 21 of the follicles 4-6 mm in diameter at 12 h. At 24 and 36 h, almost all follicles 6-9 mm in diameter, but not those 4-5 mm in diameter, expressed both P450(scc) and P450(arom) mRNA in the granulosa cells. At 48 h and thereafter, P450(scc) and P450(arom) mRNA were expressed predominantly in one healthy large follicle per cow with a few exceptions. Expression of LHr mRNA was first detected in granulosa cells at 36 h and was always found in granulosa cells of one follicle > or = 8 mm per cow with exception of one cow at 36 h (no expression) and another two cows, one each at 36 and at 84 h (expression in 2 follicles). In addition, LHr mRNA expression in the granulosa cell layer was limited to follicles that also expressed mRNAs for P450(scc) and P450(arom) in the granulosa cells. In summary, follicular recruitment in cattle was associated with expression of P450(scc) and P450(arom) mRNA within granulosa cells, and the process of follicular selection was associated with initiation of LHr mRNA expression in granulosa cells.

Ovarian follicular development in cattle is characterized by waves of growth during the prepubertal and postpartum periods and during estrous cycles. Each wave of follicular growth is characterized by recruitment of a cohort of follicles 4 to 5 mm in diameter. From the cohort, one follicle is selected for continued growth and becomes dominant. If luteolysis occurs during the growth phase of dominant follicles, final maturation and ovulation occurs. If luteolysis does not occur during the growing and maintenance phase of follicles, the fate is atresia. Changes in mRNA expression for the gonadotropin receptors (FSHr and LHr), key steroidogenic enzymes (cytochrome P450 side chain cleavage [P450scc], cytochrome P450 17alpha-hydroxylase-[P450c17], cytochrome P450 aromatase [P450arom], and 3beta-hydroxysteroid dehydrogenase [3beta-HSD]), and growth factors (IGF-I and -II) and their binding proteins (IGFBP) have been associated with different stages of follicular growth and atresia. In general, expression of mRNA for the gonadotropin receptors, steroidogenic enzymes, and steroidogenic acute regulatory protein (StAR) increase with progressive follicular development and is highest when dominant follicles approach maximum size. Expression of mRNA declines rapidly and becomes low or undetectable in atretic follicles. The IGF-I (granulosal cells) and IGF-II (thecal cells) are increased, whereas IGFBP-2 (granulosal cells) is reduced, in dominant follicles. Recruitment of a cohort of follicles is associated with initiation of expression of mRNA for P450scc and P450arom in granulosal cells. Selection of dominant follicles is associated with expression of mRNA for LHr and 3beta-HSD in granulosal cells. Thus, changes in gene expression likely are important to recruitment, selection, dominance, and atresia in ovarian follicles.

The working hypothesis was that dietary fats differing in fatty acid composition would differentially influence ovarian follicular growth. Cows (n = 27) were fed isoenergetic, isonitrogenous, and isofibrous diets containing no added fat (control; CT, n = 7) or diets supplemented with fats containing primarily saturated (SAT, n = 7), polyunsaturated (PU, n = 7), or highly polyunsaturated (HPU, n = 6) fatty acids. Coincident changes in serum lipid metabolites, insulin, and GH and the concentration of IGF-I in large and medium-sized follicles also were examined. Body weights and body condition scores remained similar for all groups throughout the study. Polyunsaturated fat increased (diet x day, P = .06) the number of medium-sized follicles on d 5 through 9 of a synchronized estrous cycle within 3 wk of onset of feeding and maximized (P < .001) this to a fourfold difference at ovariectomy after 7 wk. Fats with predominantly SAT and HPU tended (P < .10) to produce these effects after 7 wk. All fat-supplemented diets increased serum concentrations of total cholesterol (P < .05), GH (P < .05), and follicular fluid IGF-I in large follicles (P < .065) compared to CT but differentially influenced serum concentrations of insulin. Polyunsaturated fat stimulated a marked increase (P < .001) in serum insulin relative to controls within 3 wk, whereas SAT and HPU increased (P < .05) serum insulin only after 6 to 7 wk. We conclude that consumption of PU fatty acids stimulates a greater rate of ovarian follicular growth in cattle compared to CT, AT, and HPU. Future research should investigate the potential role of insulin in mediating PU effects on follicular growth.

Pregnancy-associated glycoprotein (PAG)-1 (PAG1) and pregnancy-specific protein B are either identical or closely related antigens released by trophoblast binucleate cells of placentas of cattle. Sheep and other ruminants produce similar products. There is evidence, however, that these antigens, which are related structurally to the pepsinogens and other aspartic proteinases, are not single gene products but members of an extensive family. Here, the sequential use of ammonium sulfate precipitation and Sepharose blue, anion-exchange, and cation-exchange chromatographies, as well as isoelectric elution from a Mono P column, has allowed several PAG1-related molecules to be purified from the medium after culture of explants from Day 100 sheep placentas. Each of these PAGs cross-reacted to a varying extent with a panel of three different anti-PAG1 antisera. Four of them, all of which were major secretory products of the placenta, were subjected to amino-terminal microsequencing. Although each was related to ovine (ov) PAG1, none was identical. Reverse transcription-polymerase chain reaction was then used to amplify PAG1-related cDNA from Day 100 placental RNA. Seven novel full-length cDNA, all distinct from ovPAG1, were identified from 25 cDNA selected for sequencing. Only two of these (ovPAG3 and ovPAG7) encoded polypeptides identical in sequence at their inferred amino termini to one of the PAGs (ovPAG65) purified from explant cultures. Even so, they were only 84% identical in overall sequence. The remaining five cDNA were unique. In situ hybridization analysis revealed that expression of ovPAG3 and ovPAG7, like that of ovPAG1, is confined to trophoblast binucleate cells. The data confirm that at Day 100 of pregnancy the ovine placenta produces many different PAGs, which differ considerably in sequence and immunological cross-reactivity.

Carbonic anhydrase IX (CA IX) is a transmembrane protein that has been shown to be greatly upregulated under conditions of hypoxia in many tumor cell lines. Tumor hypoxia is associated with impaired efficacy of cancer therapies making CA IX a valuable target for preclinical and diagnostic imaging. We have developed a quantitative in vivo optical imaging method for detection of CA IX as a marker of tumor hypoxia based on a near-infrared (NIR) fluorescent derivative of the CA IX inhibitor acetazolamide (AZ). The agent (HS680) showed single digit nanomolar inhibition of CA IX as well as selectivity over other CA isoforms and demonstrated up to 25-fold upregulation of fluorescent CA IX signal in hypoxic versus normoxic cells, which could be blocked by 60%–70% with unlabeled AZ. CA IX negative cell lines (HCT-116 and MDA-MB-231), as well as a non-binding control agent on CA IX positive cells, showed low fluorescent signal under both conditions. In vivo FMT imaging showed tumor accumulation and excellent tumor definition from 6–24 hours. In vivo selectivity was confirmed by pretreatment of the mice with unlabeled AZ resulting in >65% signal inhibition. HS680 tumor signal was further upregulated >2X in tumors by maintaining tumor-bearing mice in a low oxygen (8%) atmosphere. Importantly, intravenously injected HS680 signal was co-localized specifically with both CA IX antibody and pimonidazole (Pimo), and was located away from non-hypoxic regions indicated by a Hoechst stain. Thus, we have established a spatial correlation of fluorescence signal obtained by non-invasive, tomographic imaging of HS680 with regions of hypoxia and CA IX expression. These results illustrate the potential of HS680 and combined with FMT imaging to non-invasively quantify CA IX expression as a hypoxia biomarker, crucial to the study of the underlying biology of hypoxic tumors and the development and monitoring of novel anti-cancer therapies.

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Cows with ovarian follicular cysts were treated with progesterone to determine whether a reduction in LH concentrations and initiation of ovulatory follicular waves would occur. Cysts were diagnosed using transrectal ultrasonography when single follicular structures > 20 mm or multiple structures > 15 mm in diameter were present for 7 d in the presence of low progesterone concentrations. Three groups were studied: 1) cows with normal estrous cycles (CYC, n = 8); 2) cows with untreated cysts (CYST, n = 7); and 3) cows with cysts treated with two progesterone-releasing intravaginal devices (PRID, n = 8) for 9 d. Ovaries were examined with transrectal ultrasonography, and blood samples were collected daily for analysis of progesterone and FSH. Serial blood samples for determination of mean LH and LH pulse frequency were collected on d 0 (CYST and PRID cows only), 1, 5, 9, and 10. Progesterone concentrations were higher in PRID cows than in CYST cows throughout the PRID treatment period (P < .002). On d 0, LH pulse frequency was similar (P = .10) in PRID (6.6+/-.6 pulses/8 h) and CYST cows (5.1+/-.6 pulses/8 h), but mean LH tended to be higher (P = .054) on d 0 in PRID cows (2.5+/-.2 ng/mL) than in CYST cows (1.9+/-.2 ng/mL). Mean LH and LH pulse frequency decreased (P < .002) by d 1 in PRID cows (1.1+/-.2 ng/mL, 1.8+/-.6 pulses/8 h) compared with CYST cows (2.1+/-.2 ng/mL, 5.6+/-.6 pulses/8 h) and remained lower throughout most of the experimental period. The FSH concentrations were higher (P < .01) in PRID cows than in CYC and CYST cows on d 3 and 4. The increase in FSH concentrations preceded emergence of the PRID-induced follicular wave. All PRID cows and four of seven CYST cows initiated new follicular waves during the period of PRID treatment. Follicular waves were initiated later (P < .05) in CYST cows (d 5.2+/-1.7) and PRID cows (d 5.5+/-.6) than in CYC cows (d 1.8+/-.3). Cysts were smaller (P < .01) at the end of the treatment period in PRID cows compared with CYST cows. No CYST cows ovulated, but all PRID cows ovulated newly developed follicles 3 or 4 d after PRID removal. Treatment with exogenous progesterone reduced LH in cows with cysts, and this was followed by development of normal ovulatory follicles.