Daniel H. Poole

Expression and Regulation of Functional Oxytocin Receptors in Bovine T Lymphocytes

Abstract The corpus luteum (CL) produces oxytocin (OXT), which has been proposed to regulate the pulsatile release of prostaglandin F2alpha during luteolysis in ruminants. This action of OXT is mediated via oxytocin receptors (OXTRs) present on uterine epithelial cells. It is hypothesized that luteal OXT acts as a paracrine regulator of resident immune cells. In the present study, OXTR mRNA expression in bovine lymphocytes was analyzed, as well as its regulation during the estrous cycle. OXTR transcripts were observed in freshly purified bovine peripheral blood mononuclear cells and T lymphocytes. OXTR mRNA in bovine lymphocytes on Day 3 was numerically greater than but not significantly different from that of Day 19 of the estrous cycle (P = 0.091). In cultured T cells, estradiol (E2) treatment significantly increased the steady-state concentrations of OXTR mRNA, but the stimulatory effect of E2 was inhibited by the addition of progesterone (P4). Each of the major T cell subsets (CD4+, CD8+, and gamma delta+) expressed OXTR mRNA, with no significant difference in expression among them. Western blot analyses demonstrated the presence of the bovine OXTR protein at about 45 kDa in lymphocytes, as well as expression of the 14-kDa precursor of OXT. When lymphocytes were treated with OXT, intracellular concentrations of calcium ([Ca2+]i) were rapidly and dramatically increased. This study demonstrated that bovine lymphocytes express OXTRs and that this expression can be regulated in a steroid-dependent manner. Furthermore, OXT elicited a functional [Ca2+]i response in T lymphocytes, supporting the possibility that OXT within the CL could act as a paracrine or autocrine regulator of resident T lymphocytes.

Luteal Microenvironment Directs Resident T Lymphocyte Function in Cows

ABSTRACT The corpus luteum (CL) is a transient endocrine organ composed of a heterogeneous mixture of cells. Functional interactions exist between peripheral T cells and luteal cells in vitro; however, the precise role of resident T cells (RTC) remains unknown. The goals of the present study were to isolate RTC from within the CL and determine if alteration of luteal function resulted in changes in RTC phenotypes. Functional lymphocyte phenotypes identified in the bovine CL by using quantitative flow cytometric analysis were clearly different from those in the peripheral circulation. The proportion of CD8+ RTC was greater than CD4+ RTC. These proportions were opposite in peripheral blood. The proportion of γδ+ lymphocytes was not different in the CL compared to that in peripheral blood nor was it altered during luteal regression. There was a significant increase in CD8αα+ and γδ+CD8αα+ RTC during luteal regression. The proportion of FOXP3+ lymphocytes in the CL was greater than that isolated from peripheral blood, and this proportion of lymphocytes was dramatically reduced by induction of luteolysis. Within the CL of early pregnancy, there was an increase in the CD8αβ+ and γδ+CD8αβ+ populations compared to those in the CL of nonpregnant animals. Based on these data, we concluded that the functional state of the CL creates a microenvironment that regulates the recruitment of or differentiation into specific lymphocyte types. Understanding the interactions between steroidogenic cells and ovarian lymphocytes within CL will not only enhance understanding of reproductive function but may provide vital clues to lymphocyte regulation within tissues.

Progesterone effects on lymphocytes may be mediated by membrane progesterone receptors

Luteal cell-induced proliferation of T lymphocytes devoid of the nuclear progesterone receptor (PGR) is inhibited by progesterone. Functional effects of progesterone on bovine lymphocytes and the expression of membrane progesterone receptors (mPRs) alpha (PAQR7), beta (PAQR8), gamma (PAQR5), and progesterone receptor membrane component 1 (PGRMC1) mRNA were analyzed in corpus luteum (CL) and lymphocytes. Progesterone and a cell-impermeable progesterone conjugate caused a dose-dependent decrease in IL2 receptor α-subunit (IL2RA) mRNA and an increase in interleukin 2 (IL2) mRNA concentrations in cultured PBMCs. In luteal tissues, concentrations of PAQR7 and PAQR8 mRNA were lower in CL collected on day 11 compared with day 18, whereas PGRMC1 and PGR mRNA were greater on day 11 than on day 18. The mRNA of all three PAQRs and PGRMC1 were detected in bovine T lymphocytes, but not in B cells/monocytes. Progesterone increased intracellular Ca(++) and reduced the phosphorylation of zeta-chain-associated protein kinase 70 (Zap70). A specific, saturable, and single progesterone binding site with a steroid specificity characteristic of mPRs was demonstrated by saturation and competitive binding assays using T lymphocyte membranes, and PAQR7 receptors were localized on the plasma membranes by immunofluorescence. Thus, progesterone induces specific and rapid functional effects on T lymphocytes in the absence of PGR. The mPRs are potential intermediaries of the cell-surface actions of progesterone because they are expressed in lymphocytes, the actions of progesterone are mimicked by a cell-impermeable form of progesterone, and specific, saturable progesterone binding, which is characteristic of mPRs, is present on lymphocyte membranes.

Low peripheral progesterone and late embryonic/early fetal loss in suckled beef and lactating dairy cows

Pregnancy failure during placentation in lactating dairy cows was associated with low concentrations of serum progesterone. Beef cows have greater serum progesterone and less pregnancy failure. Experiment 1 determined that reduction of serum progesterone affected late embryonic/early fetal loss in suckled beef cows. Cows (n=40) received progesterone from two new or used controlled internal drug releasing devices, replaced every 5d, beginning on Day 28 of gestation (mating=Day 0); CL were enucleated on Day 29. Retention of pregnancy was 77% in treated cows and 97% in 78 control cows (P<0.05). Experiment 2 determined how pregnant, lactating dairy cows with high or low progesterone concentrations during Days 28-34 differed in luteal function or in serum progesterone during replacement therapy. Luteal tissue from such cows was assayed for progesterone and expression of mRNA for genes of endothelin and prostaglandin (PG) systems. Secretion of progesterone and prostaglandins by dispersed luteal cells was determined during incubation with LH, endothelin-1, or arachidonic acid. Neither luteal progesterone nor mRNAs for endothelin or prostaglandin systems differed. Endothelin-1 inhibited secretion of progesterone more (P<0.05) in luteal cells from cows with low versus high serum progesterone, when incubated with arachidonic acid. Secretion of prostaglandin F(2)alpha was increased and that of 6-keto-PGF(1)alpha decreased by endothelin-1 in vitro. Serum progesterone during replacement was lower (P<0.05) for cows with low than high serum progesterone at lutectomy. Thus, clearance, more than luteal production, determined peripheral progesterone in pregnant, lactating dairy cows.

Bone morphogenetic protein 6 promotes FSH receptor and anti-Müllerian hormone mRNA expression in granulosa cells from hen prehierarchal follicles

A growing body of literature provides evidence of a prominent role for bone morphogenetic proteins (BMPs) in regulating various stages of ovarian follicle development. Several actions for BMP6 have been previously reported in the hen ovary, yet only within postselection (preovulatory) follicles. The initial hypothesis tested herein is that BMP6 increases FSH receptor (FSHR) mRNA expression within the granulosa layer of prehierarchal (6-8 mm) follicles (6-8 GC). BMP6 mRNA is expressed at higher levels within undifferentiated (1-8 mm) follicles compared with selected (≥9 mm) follicles. Recombinant human (rh) BMP6 initiates SMAD1, 5, 8 signaling in cultured 6-8 GC and promotes FSHR mRNA expression in a dose-related fashion. In addition, a 21 h preculture with rhBMP6 followed by a 3 h challenge with FSH increases cAMP accumulation, STAR (StAR) expression, and progesterone production. Interestingly, rhBMP6 also increases expression of anti-Müllerian hormone (AMH) mRNA in cultured 6-8 GC. This related BMP family member has previously been implicated in negatively regulating FSH responsiveness during follicle development. Considering these data, we propose that among the paracrine and/or autocrine actions of BMP6 within prehierarchal follicles is the maintenance of both FSHR and AMH mRNA expression. We predict that before follicle selection, one action of AMH within granulosa cells from 6 to 8 mm follicles is to help suppress FSHR signaling and prevent premature granulosa cell differentiation. At the time of selection, we speculate that the yet undefined signal directly responsible for selection initiates FSH responsiveness. As a result, FSH signaling suppresses AMH expression and initiates the differentiation of granulosa within the selected follicle.

Adipogenic differentiation state-specific gene expression as related to bovine carcass adiposity

Genetic regulation of the site of fat deposition is not well defined. The objective of this study was to investigate adipogenic differentiation state-specific gene expression in feedlot cattle (>75% Angus; <25% Simmental parentage) of varying adipose accretion patterns. Four groups of 4 steers were selected via ultrasound for the following adipose tissue characteristics: low subcutaneous-low intramuscular (LSQ-LIM), low subcutaneous-high intramuscular (LSQ-HIM), high subcutaneous-low intramuscular (HSQ-LIM), and high subcutaneous-high intramuscular (HSQ-HIM). Adipose tissue from the subcutaneous (SQ) and intramuscular (IM) depots was collected at slaughter. The relative expression of adipogenic genes was evaluated using quantitative PCR. Data were analyzed using the mixed model of SAS, and gene expression data were analyzed using covariate analysis with ribosomal protein L19 as the covariate. No interactions (P > 0.10) were observed between IM and SQ adipose tissue depots for any of the variables measured. Therefore, only the main effects of high and low accretion within a depot and the effects of depot are reported. Steers with LIM had smaller mean diameter IM adipocytes (P < 0.001) than HIM steers. Steers with HSQ had larger mean diameter SQ adipocytes (P < 0.001) than LSQ. However, there were no differences (P > 0.10) in any of the genes measured due to high or low adipose accretion. Preadipogenic delta-like kinase1 mRNA was greater in the IM than the SQ adipose tissue; conversely, differentiating and adipogenic genes, lipoprotein lipase, PPARγ, fatty acid synthetase, and fatty acid binding protein 4 were greater (P < 0.001) in the SQ than the IM depot. Intramuscular adipocytes were smaller than SQ adipocytes and had greater expression of the preadipogenic gene, indicating that more hyperplasia was occurring. Meanwhile, SQ adipose tissue contained much larger (P < 0.001) adipocytes that had a greater expression (P < 0.001) of differentiating and adipogenic genes than did the IM adipose tissue, indicating more cells were undergoing differentiation and hypertrophy. Adipogenic differentiation state-specific gene expression was not different in cattle with various phenotypes, but adipogenesis in the SQ and IM adipose tissues seems to occur independently.

Pregnancy outcome in dairy and beef cattle after artificial insemination and treatment with seminal plasma or transforming growth factor beta-1

Reduced capability of the uterus to support pregnancy in the absence of its interaction with secretions from male accessory glands has been demonstrated in rodents and to some extent in pigs. However, in cattle, the role of postmating inflammatory response on pregnancy success has not been studied. The current study examined the influence of uterine presensitization with seminal antigens at breeding on pregnancy outcome in cows. Lactating beef (n=1090) and dairy (n=800) cows received 0.5 mL seminal plasma (SP), 40 ng recombinant human transforming growth factor-beta1 (rhTGF-beta1), or 0.5 mL bovine serum albumin (BSA), or were left untreated before or at insemination. Semen was deposited into the anterior cervix using a second insemination gun. Pregnancy was diagnosed at 35 to 40 d postinsemination by transrectal ultrasonography or from records of calves born the subsequent calving season. Pregnancy rates in beef cows did not differ among treatments but differed among trials (69.8%, 52.5% vs. 40.3%; P<0.05). In trials where average pregnancy rates were below 50%, treatments with TGF-beta1 but not SP tended (P<0.07) to increase pregnancy rates in beef cows. In dairy cows, SP and TGF-beta1 improved pregnancy outcome by 10 percentage points, but these increments did not achieve statistical significance. In conclusion, this study did not find any conclusive evidence for the effect of TGF-beta1 or seminal plasma on pregnancy outcome in lactating dairy or beef cows but realized marginal improvements when pregnancy rates were below 50% (compromised fertility).

Expression and regulation of secreted phosphoprotein 1 in the bovine corpus luteum and effects on T lymphocyte chemotaxis

Secreted phosphoprotein 1 (SPP1) in the bovine corpus luteum (CL) regulates cell function during the transitional periods of luteinization and luteal regression. The objectives were to i) characterize SPP1 expression in the CL throughout the estrous cycle, ii) determine factors that regulate SPP1 expression in luteal cells, and iii) examine the role of SPP1 in lymphocyte chemotaxis, proliferation, and function. SPP1 mRNA was greater in fully functional (d10) CL and late cycle (d18) CL compared with developing (d4) CL. Additionally, SPP1 mRNA increased within 1 h and remained elevated 4 and 8 h following induction of luteolysis with prostaglandin (PG)F2α. Expression of the SPP1 receptor, β3 integrin, was not different throughout the estrous cycle but decreased following induction of luteolysis. Expression of CD44 increased during the estrous cycle but did not change during luteal regression. In cultured luteal cells, SPP1 mRNA was upregulated by PGF2α and/or tumor necrosis factor α. Western blots revealed the presence of both full-length SPP1 and multiple cleavage products in cultured luteal cells and luteal tissue. Depletion of endogenous SPP1 did not hinder luteal cell-induced lymphocyte proliferation or lymphocyte phenotype but did inhibit lymphocyte migration toward luteal cells. Based on these data, it is concluded that SPP1 is initially activated to establish and maintain cellular interactions between steroidogenic and nonsteroidogenic cells during the development of the CL. Upon induction of luteolysis, SPP1 serves as a signaling molecule to recruit or activate immune cells to facilitate luteal regression and tissue degradation.

Asymptotic distribution of the numbers of vertices and arcs of the giant strong component in sparse random digraphs

Two models of a random digraph on n vertices, D(n; Prob(arc) = p) and D(n; number of arcs = m) are studied. In 1990, Karp for D(n;p) and independently T. Luczak for D(n;m = cn) proved that for c > 1, with probability tending to 1, there is an unique strong component of size of order n. Karp showed, in fact, that the giant component has likely size asymptotic to n 2 , where = (c) is the unique positive root of 1 = e c . In this paper we prove that, for both random digraphs, the joint distribution of the number of vertices and number of arcs in the giant strong component is asymptotically Gaussian with the same mean vector n (c), (c) := ( 2 ;c 2 ) and two distinct 2 2 covariance matrices, nB(c) and n B(c) +c( 0 (c)) T ( 0 (c))) . To this end, we introduce and analyze a randomized deletion process which determines the directed (1; 1)-core, the maximal digraph with minimum in-degree and out-degree at least 1. This (1; 1)-core contains all nontrivial strong components. However, we show that the likely numbers of peripheral vertices and arcs in the (1; 1)-core, those outside the largest strong component, are of polylog order, thus dwarfed by anticipated uctuations, on the scale of n 1=2 , of the giant component parameters. By approximating the likely realization of the deletion algorithm with a deterministic trajectory, we obtain our main result via exponential supermartingales and Fourier-based techniques.

Effects of service sire on prenatal mortality and prolificacy in ewes.

Ability to select service sires that minimize partial or complete losses of pregnancy could have major economic impacts in sheep production systems. This study tested the null hypothesis that survival of potential progeny did not vary with breed type of service sire or among individual rams. Data included 980 ewes on 10 farms; each ewe was pregnant to 1 of 67 rams of 12 breeds. Number of conceptuses was estimated once during pregnancy by ultrasonography, either transrectal (embryos) or transabdominal (fetuses), and was compared with number of lambs born to estimate losses. Data were examined first for number of lambs born and second for documented losses. Individual service sires affected number born (P < 0.001), which varied from 0.70 to 2.45 lambs per pregnant ewe. The main effects of breed type on lambs born were not significant, but breed types of both service sires (P < 0.0002) and ewes (P < 0.001) interacted with diagnosed number of conceptuses. Lambs born varied with ewe age (P < 0.0001) and among farms (P < 0.0001), and statistically, farms interacted with number of diagnosed conceptuses (P < 0.0001); season had no effect. In documented losses, there were both main effects of individual service sire and a service sire × number of diagnosed embryos interaction (P < 0.005). Thus, ewes bred to some rams were more apt to lose single pregnancies, whereas ewes bred to other rams were more apt to lose 1 or more embryos or fetuses from multiple pregnancies. Breed type of service sire affected (P < 0.05) prenatal death. Complete losses of single conceptuses tended to be greater in ewes bred to black-faced or hair-type rams (service sire breed type × number of diagnosed conceptuses; P < 0.09). Breed type of ewes also varied in incidence of complete losses (P < 0.05); hair-type ewes (46%) lost more (P < 0.02) documented conceptuses from examination to birth than black-faced (27%), white-faced (20%), or dairy-type (25%) ewes. Greater losses of singles than of multiples occurred in black-faced (37% vs. 18%) and hair-type (64% vs. 27%) ewes than in other breeds (ewe breed type × number of conceptuses; P < 0.03) per ewe. Surprisingly, purebred conceptuses were lost less often (24%) than crossbreds (36.4%; P < 0.002). Selection of rams based on records of prenatal losses in ewes they serviced may be a method to decrease embryonic and fetal wastage. However, further study to determine repeatability of differences among service sires from year to year will be required.