R. S. Youngquist

Follicular Dominance in Cattle Is Associated With Divergent Patterns of Ovarian Gene Expression for Insulin-Like Growth Factor (IGF)-I, IGF-II, and IGF Binding Protein-2 in Dominant and Subordinate Follicles

A decrease in insulin-like growth factor (IGF) binding protein (BP) amount occurs within the follicular fluid of dominant ovarian follicles. At the same time, concentrations of follicular fluid IGF-I do not change. The mRNA for IGF-I, IGF-II, IGFBP-2, and IGFBP-3, in dominant and subordinate follicles were measured to determine if changes in IGF or IGFBP gene expression are associated with follicular dominance. Heifers were ovariectomized during a follicular wave, either during early-dominance (emerging dominant follicle, 9 mm diameter) or mid-dominance (established dominant follicle, 14-16 mm diameter). Follicles were classified as either dominant (DF), subordinate (SF), or not-recruited (NRF; small antral follicles). mRNA was localized by in situ hybridization and measured by image analyses. The IGF-I mRNA (granulosa cells) was greatest in DF and increased in DF, SF, and NRF from early- to mid-dominance. Likewise, IGF-II mRNA (theca cells) was greatest in DF compared with SF or NRF. The IGFBP-2 mRNA (granulosa cells), however, was nearly undetectable in DF, whereas adjacent SF expressed abundant IGFBP-2 mRNA. The NRF were not uniform in their IGFBP-2 expression because only 5 of 13 NRF had IGFBP-2 mRNA. The IGFBP-3 mRNA (granulosa cells) was found only in two NRF, suggesting that local synthesis is not a predominant source of follicular fluid IGFBP-3. These data show that changes in gene expression for IGFBP-2 are opposite to those for IGF-I or IGF-II. Increased IGF-I and IGF-II mRNA and decreased IGFBP-2 mRNA within the DF may be one mechanism leading to follicular dominance. The opposite pattern of IGFBP-2 gene expression in SF and some NRF may lead to follicular atresia.

Dominant Bovine Ovarian Follicular Cysts Express Increased Levels of Messenger RNAs for Luteinizing Hormone Receptor and 3β-Hydroxysteroid Dehydrogenase Δ4,Δ5 Isomerase Compared to Normal Dominant Follicles

Abstract The objective was to compare ovarian steroids and expression of mRNAs encoding cytochrome P450 side-chain cleavage, cytochrome P450 17α-hydroxylase, cytochrome P450 aromatase, 3β-hydroxysteroid dehydrogenase Δ4,Δ5 isomerase, LH, and FSH receptors and estrogen receptor-β in ovaries of cows with dominant and nondominant ovarian follicular cysts and in normal dominant follicles. Estradiol-17β, progesterone, and androstenedione concentrations were determined in follicular fluid using specific RIAs. Dominant cysts were larger than young cysts or dominant follicles, whereas nondominant cysts were intermediate. Estradiol-17β (ng/ml) and total steroids (ng/follicle) were higher in dominant cysts than in dominant follicles. Expression of LH receptor and 3β-hydroxysteroid dehydrogenase mRNAs was higher in granulosa cells of dominant cysts than in dominant follicles. Nondominant cysts had higher follicular concentrations of progesterone, lower estradiol-17β concentrations, and lower expression of steroidogenic enzyme, gonadotropin receptor, and estrogen receptor-β mRNAs than other groups. In summary, increased expression of LH receptor and 3β-hydroxysteroid dehydrogenase mRNAs in granulosa and increased follicular estradiol-17β concentrations were associated with dominant cysts compared to dominant follicles. Study of cysts at known developmental stages is useful in identifying alterations in follicular steroidogenesis.

Concentrations of nonesterified fatty acids and glucose in blood of periparturient dairy cows are indicative of pregnancy success at first insemination

Greater blood concentrations of nonesterified fatty acids (NEFA) and lesser blood concentrations of glucose are indicative of the normal process of nutrient partitioning that occurs in early postpartum dairy cows. The objective was to determine the relationship between blood NEFA and glucose concentrations and subsequent conception at first insemination in postpartum dairy cows. Holstein (n=148) and Guernsey (n=8) dairy cows were blood sampled at approximately d 10, 7, and 3 prepartum, on the day of calving and 3, 7, 14, and 21 d postpartum for measurement of NEFA and glucose concentrations. Serum and plasma were harvested and used for measurement of NEFA and glucose concentrations, respectively. Cows were given a presynchronization treatment (2 injections of PGF(2α) 14 d apart) with the second PGF(2α) injection occurring 14 d before the initiation of the timed AI (TAI) protocol. Blood for determination of progesterone concentrations was collected at each presynchronization injection and at the initiation of the TAI protocol that was used for first insemination (74±7 d postpartum). Cows were considered noncycling if serum progesterone concentrations at the 2 presynchronization PGF(2α) injections (d 37 and 51±7 postpartum) and at the initiation of the TAI protocol (d 65±7 postpartum) were ≤1 ng/mL, and there was no indication of ovulation or presence of a corpus luteum by ultrasound examination at the initiation of the TAI protocol. Pregnancy was determined at 33 d and again at 61 d after first insemination by using ultrasound. Across all days, serum NEFA and plasma glucose concentrations were not different between cows that ovulated before the initiation of the TAI program (cycling) compared with those that did not ovulate (noncycling). Serum NEFA concentrations, however, were less and plasma glucose concentrations were greater during the early postpartum period for cows that subsequently became pregnant at first insemination compared with those that failed to become pregnant. Logistic regressions were used to predict the probability of pregnancy based on NEFA and glucose concentrations from individual days. The prediction with the greatest likelihood ratio was for d 3 postpartum NEFA and glucose concentrations. Nutritional status during the early postpartum period (within 1 wk after calving), as indicated by blood NEFA and glucose concentrations, may affect subsequent fertility by a mechanism that is independent from interval to first ovulation.

Plasma gh, igf-i, and conception rate in cattle treated with low doses of recombinant bovine gh

Blood and uterine concentrations of GH and insulin-like growth factor (IGF)-I are correlated with improved fertility in cattle. We tested incremental doses of a 14-d sustained release recombinant bovine GH (rbGH) to increase blood GH and IGF-I (Experiments 1 and 2). Conception rate after administration of an optimized rbGH dose was also tested (Experiment 3). In Experiment 1, lactating Holstein cows (n = 18) were randomly assigned to receive 0 (n = 5), 100 (n = 5), 200 (n = 5), or 500 (n = 3) mg sc rbGH. Increasing the doses of rbGH was associated with increased serum concentrations of GH and IGF-I. The 100- and 200-mg doses caused an IGF-I release that was below and above, respectively, the perceived optimum response. Therefore, Experiment 2 was designed to test a rbGH dose (167 mg), which was intermediate to the doses tested in Experiment 1. Lactating and nonlactating postpartum beef cows were treated with 0 (n = 9) or 167 (n = 9) mg rbGH at insemination. Plasma concentrations of GH and IGF-I were greater in rbGH-treated cows than in controls. Lactating cows had initial IGF-I concentrations that were lower than nonlactating cows. The 167-mg dose of rbGH increased plasma IGF-I concentrations in lactating cows to the levels of those of nonlactating cows. In Experiment 3, cows and heifers were administered either 0 or 167 mg rbGH at insemination. The conception rate for rbGH-treated and control cows was 54.4 and 49.5% (n = 617), and 46.0 and 46.3% for heifers (n = 1123), respectively. Herd (P<0.01) and parity (P<0.01) affected conception rate, but conception rates for rbGH and control cattle were similar. In summary, low doses of rbGH increased blood GH and restored blood IGF-I concentrations in lactating cows to those of nonlactating cows, but the conception rate in cows and heifers was not affected by administration of 14-d sustained-release rbGH at insemination.

Luteinizing hormone response to estradiol benzoate in cows postpartum and cows with ovarian cysts

Twenty-seven dairy cows were evenly assigned to one of three groups and given an intramuscular injection of 2 mg estradiol benzoate. Cows in group 1 were greater than 30 days postpartum at treatment and had been diagnosed via rectal palpation to have ovarian cysts. Cows in groups 2 and 3 were 12 to 14 and 30 to 40 days postpartum, respectively. Blood plasma was collected from all cows before treatment and then every three hours for 36 hours post-treatment. Concentrations of LH, estradiol-17 beta and progesterone in plasma were determined by radioimmunoassay. Four, zero and five cows in groups 1, 2 and 3, respectively, had concentrations of progesterone greater than 1.0 ng/ml before estradiol benzoate treatment. None of these cows had a peak LH release greater than 5 ng/ml following estradiol benzoate treatment. The numbers of cows with progesterone concentrations less than 1 ng/ml that released LH (>5 ng/ml) in response to estradiol benzoate were 3 of 5, 3 of 9, and 4 of 4 for groups 1, 2, and 3, respectively; the proportion for group 3 was higher (P<.05) than for group 2. Of the cows that released LH, mean peak LH concentrations were 33.3+/-5.4, 14.8+/-7.2 and 24.6+/-9.8 ng/ml for groups 1, 2 and 3, respectively, and the duration of the LH increase was 8.0+/-1.0, 8.0+/-2.0 and 13.0+/-4.0 hours. The time from estradiol benzoate treatment to peak LH release for cows with ovarian cysts (25+/-2 hours) was delayed (P<.05) compared with that for cows 30 to 40 days postpartum without ovarian cysts (16+/-1 hour). In summary, responsiveness to estradiol benzoate is regained between 2 to 4 weeks postpartum in most cows. In addition, some cows with ovarian cysts can release LH in response to estradiol benzoate, but peak LH release is delayed compared to cows at a comparable stage postpartum without ovarian cysts.

Concentrations of steroids and expression of messenger RNA for steroidogenic enzymes and gonadotropin receptors in bovine ovarian follicles of first and second waves and changes in second wave follicles after pulsatile LH infusion

The objectives were to compare expression of mRNA for cytochrome P450 cholesterol side-chain cleavage (P450scc), cytochrome P450 17alpha-hydroxylase (P450c17), cytochrome P450 aromatase (P450arom), 3beta-hydroxysteroid dehydrogenase Delta(4), Delta(5) isomerase (3beta-HSD), FSH receptor (FSHr) and LH receptor (LHr) in bovine ovarian follicles of the first and second waves of the bovine oestrous cycle and to determine if LH infusion changes growth, steroidogenesis and gene expression in second wave follicles. Transrectal ultrasonography was used to examine follicular size changes during the oestrous cycle in non-lactating Holstein cows (n=31). Saline or purified bovine LH was infused intravenously into cows at emergence of follicular waves for 2 or 4 days using a computer-controlled syringe pump (n=5-6 per treatment). Treatments were: wave 1, saline (W1S); wave 2, saline (W2S) or LH (25 microg/h; W2LH). During infusion, blood samples were collected at 12min intervals for 8h via i.v. catheters for measurement of serum LH concentrations. Ovaries were removed from cows on days 2 or 4 after emergence of follicular waves. Follicles were frozen and stored at -80 degrees C. Follicular fluid (FF, 50 microl) was collected for determination of progesterone (P4), oestradiol-17beta (E2) and androstenedione (A4) concentrations. Frozen sections (14 microm) were used for in situ hybridization to measure expression of mRNA (% pixel intensity) for P450scc, P450c17, P450arom, 3beta-HSD, FSHr, and LHr. LH infusion resulted in a serum LH pattern (high frequency) similar to the early luteal phase. There were no significant differences in size of follicles among the three treatment groups. Follicular fluid concentrations of E2 and A4 in W2S were lower than those of W1S on day 2 of a follicular wave. LH infusion into cows during the midluteal phase increased follicular fluid E2 and A4 concentrations in second wave follicles on day 2 of a follicular wave (W2LH) compared to those of W2S. The increase in follicular fluid E2 on day 2 in wave 2 follicles after LH infusion occurred possibly through an increase in mRNA expression of P450c17 and 3beta-HSD. In conclusion, follicular fluid concentrations of E2 and A4 were lower in W2S than in W1S and E2 and A4 concentrations were restored by infusion of LH in W2LH with an increase in mRNA expression of P450c17 and 3beta-HSD.

Ontogenies of messenger RNA encoding tissue inhibitor of metalloproteinases 1 and 2 within bovine periovulatory follicles and luteal tissue

Tissue inhibitors of metalloproteinases 1 and 2 (TIMP-1 and TIMP-2) are important regulators of extracellular matrix remodeling and also possess growth factor activity. The objective of these studies was to characterize TIMP-1 and TIMP-2 mRNA expression by bovine periovulatory follicles/ corpora hemorrhagica (Experiment 1) and luteal tissue (Experiment 2). In Experiment 1, beef heifers (n = 27) were ovariectomized at-16 (n = 6), 0 (n = 5), 8 (n = 3), 16 (n = 4), 24 (n = 4), or 48 (n = 5) hr relative to a gonadotropin-releasing hormone induced gonadotropin surge (40 hr after prostaglandin F2 alpha-induced luteolysis). Total cellular RNA was isolated from the large steroidogenically active follicle or corpus hemorrhagicum obtained from each animal, and the expression of TIMP-1 and TIMP-2 mRNA was subsequently examined by northern and dot blot analysis. The expression of TIMP-1 or TIMP-2 mRNa did not differ in preovulatory follicles collected at -16 vs. 0 hr. Concentrations of both TIMP-1 and TIMP-2 mRNA (picograms per microgram of tissue DNA) were increased (P < 0.05) at 8 hr postgonadotropin surge, had declined to presurge levels by 24 hr (P < 0.05), and were increased (P < 0.05) in corpora hemorrhagica collected at 48 hr after a gonadotropin surge. In Experiment 2, corpora lutea were collected from beef heifers on Days 4, 10, 15 (n = 4 each), or 19 (n = 3) postestrus (Day 0 = estrus). Concentrations of TIMP-1 mRNA (picograms per microgram of tissue DNA) were greater in corpora lutea collected on Day 4 (P < 0.05) vs. Day 10, 15, or 19. Concentrations of TIMP-2 mRNA increased (P < 0.05) from Day 4 to 15 and decreased (P < 0.05) by Day 19. We conclude that: 1) during the periovulatory period, the ontogenies of TIMP-1 and TIMP-2 mRNA expression are similar, whereas 2) during luteal phase, TIMP-1 mRNA expression is maximal during the early luteal phase, whereas concentrations of TIMP-2 mRNA peak during the midluteal phase. TIMP-1 and TIMP-2 may play important roles in the regulation of extracellular matrix remodeling during the periovulatory period and the subsequent luteal phase.

Molecular Mechanisms Regulating Bovine Ovarian Follicular Selection

Transcription profiling of ovarian follicles. Understanding the mechanisms by which a single follicle is selected for further ovulation is important to control fertility in mammals. However, development of new treatments is limited by our poor understanding of molecular mechanisms regulating follicular selection. Our hypothesis is that genes involved in the control of cell proliferation and apoptosis are differentially regulated during follicular selection. Our objective was to identify these new genes. Bovine follicles were collected and gene expression levels were measured using microarrays. First, follicles were allocated to three groups, according to the time spent from the initiation of follicular wave to surgery (24 H, 36 H, and 48–60 H). Fifty‐seven genes are differentially expressed at a false discovery rate of 5%. These genes are involved in the control of lipid metabolism (P‐value = 0.0005), cell proliferation (0.007), cell death (0.003), cell morphology (0.003), and immune response (0.003). Follicles were also grouped into four categories, according to the expected time of deviation (early deviation; 8 mm, mid‐deviation; 8.5 mm, late deviation; 9 mm, dominant follicles; ≥10 mm). One hundred and twenty eight genes are differentially expressed between these four groups, including genes involved in cell proliferation (0.00002), cell death (0.0006), cell‐to‐cell signaling (0.003), cell morphology (0.003), lipid metabolism (0.0004), and immune response (0.00007). The expression levels of 10 genes were confirmed using quantitative real time PCR. As expected, we identified new differentially regulated genes involved in the control of cell growth and apoptosis. We also discovered a potential role for immune cells, and in particular macrophages, in follicular selection. Mol. Reprod. Dev. 76: 351–366, 2009.

Luteinizing hormone receptor concentrations, adenylate cyclase activity and phosphodiesterase activity of bovine corpora lutea: Comparison of short and normal estrous cycles☆

Abstract Concentrations of unoccupied and occupied luteinizing hormone (LH) receptors, basal and agonist-activated adenylate cyclase activities and phosphodiesterase activity of corpora lutea from seventeen postpartum beef cows anticipated to have a short or normal luteal lifespan were compared. Calves were weaned 26 to 33 days postpartum and the cows assigned to groups 1 or 2. In group 1 (n=8), corpora lutea were removed on day 5 (day 0 = estrus) following the first and third estrus. In group 2 (n=9), lutectomy occurred on day 5 following the third estrus. The length of the first estrous cycle ranged from 7 to 12 days, ( x ± SD =8.1 ± 1.6 days ) in eight of nine cows in group 2 and the mean ( x ± SE ) length of the second estrous cycle for groups 1 and 2 was 19.6 ± .2 and 20.9 ± .4 days, respectively. Luteal weight, luteal progesterone concentration, plasma progesterone concentration, luteal unoccupied and occupied LH receptor concentrations, and luteal adenylate cyclase (basal and agonist-activated) activities were similar (P>.05) on day 5 of the first (group 1) and third (groups 1 and 2) estrous cycles. The preceding plasma and luteal measurements were similar (P>.05) on day 5 of the third estrous cycle for groups 1 and 2. Luteal phosphodiesterase activity was similar (P>.05) for groups 1 and 2 on day 5 of the third estrous cycle, but higher (P

Follicular growth and endocrine patterns of prepuberal heifers administered bovine follicular fluid and (or) follicle stimulating hormone

Two experiments were conducted to determine if charcoal-extracted follicular fluid (CFF) from bovine ovaries inhibits FSH-induced follicular development in intact prepuberal heifers. In Exp. 1, thirty prepuberal heifers were assigned to saline, FSH or CFF/FSH treatment groups. In Exp. 2, thirty-two prepuberal heifers were assigned to four treatment groups (Saline, CFF, FSH, CFF/FSH) in a 2×2 factorial arrangement. In both studies, heifers were injected (i.v.) every 8 h for 88 h with CFF (8 ml, Exp. 1; 20 ml, Exp. 2) or saline. Follicle stimulating hormone (3.3 mg with 14% LH, Exp. 1; 3.3 mg with <1% LH, Exp. 2) was injected (i.m.) every 8 h starting 24 h after the initiation of CFF injections and continuing until termination of CFF administration. Plasma samples were collected via jugular venipuncture at 8 h intervals from just prior to (Exp. 1) or 48 h before (Exp. 2) CFF treatment until 96 h after, at which time both ovaries were removed. In both experiments total ovarian and follicular fluid weights increased (P<0.05) following FSH treatment. However, total number of follicles was similar among treatments, FSH induced a shift (P<0.05) from small (≤3 mm) to medium (7 to 9 mm) or large (10 to 13 mm) follicles. None of the above indices of FSH-induced follicular growth were affected (inhibited) by CFF. In summary, CFF did not inhibit FSH-induced follicular development in intact prepuberal heifers.