Joy L. Pate

Effects of propylene glycol drenching on energy balance, plasma glucose, plasma insulin, ovarian function and conception in dairy cows

We postulated that daily drenching of propylene glycol to cows in early lactation would increase plasma glucose and insulin concentrations and improve fertility in postpartum cows. Thirty-six Holstein cows were assigned to treatment or control groups. Each treatment cow was given 500 ml of propylene glycol by drenching daily from 7 to 42 days of lactation. Blood samples for glucose, insulin, nonesterified fatty acids (NEFA), and plasma urea N were collected at 0, 30, and 90 min postdrenching once weekly during 1-6 weeks. Blood samples were collected for progesterone analysis and cows were palpated three times per week until 11 weeks to assess ovarian status. Propylene glycol did not affect dry matter intake (DMI), milk yield or energy balance in treatment cows. After drenching, propylene glycol increased (P<0.01) plasma glucose and insulin and decreased (P<0.01) NEFA; plasma urea N of the treatment group tended (P=0.07) to be higher than that of the control group through 90 min. Days to first service, days open, and services per conception were not different between groups. Conception rates to first insemination were 33% in the control group and 57% in treated cows, but these were not significantly different. First ovulation of treatment cows occurred earlier than that of control cows (32.3 versus 44.5 days, P=0.06) and the length of the first luteal phase was longer in treated cows (13.1 versus 7.3 days, P<0.05). These data are consistent with the hypothesis that insulin is important for normal ovarian function. During negative energy balance, treatment with propylene glycol, which induced small increases in plasma concentrations of insulin, prevented the short luteal phase characteristic of the first estrous cycle in control cows.

Effects of serum and lipoproteins on steroidogenesis in cultured bovine luteal cells

The effects of serum and lipoproteins on the function of bovine luteal cells in tissue culture were examined. Corpora lutea from regularly cycling dairy cows were dissociated with collagenase and cultured in Hams F-12 medium with or without serum. The serum-free medium was supplemented with insulin, transferrin and hydrocortisone. Addition of LH to the serum-containing medium did not increase progesterone (P4) production. When the luteal cells were cultured in serum-free medium. LH produced a dose-dependent increase (P less than 0.001) in P4 production during the first 24 h of culture. The responsiveness of the cells to LH then gradually declined, and remained low until days 9-11 of culture, at which time the cells regained their ability to respond to LH. The luteal cells were responsive to dibutyryl cAMP in both serum-containing and serum-free medium. Serum lipoproteins (low and high density) were able to produce a 150-260% increase in progesterone production by the luteal cells cultured in serum-free medium. The results indicate that the presence of serum in the cell culture medium inhibits the responsiveness of luteal cells to LH at a step prior to the increase in cellular cAMP, and that serum lipoproteins can be used to increase progesterone production by cultured bovine luteal cells.

Regulation of prostaglandin synthesis by progesterone in the bovine corpus luteum.

The objective of the present study was to investigate the influence of progesterone on prostaglandin synthesis by the corpus luteum (CL). Corpora lutea were obtained from dairy cows on days 4, 6, 10, and 18 of the estrous cycle, dissociated, and placed in serum-free culture. The addition of luteinizing hormone (LH) resulted in a slight, but non-significant (p greater than 0.05), increase in levels of 6-keto-PGF1 alpha, and had no effect on PGF2 alpha. Progesterone treatment caused a significant, dose-dependent decrease in both PGF2 alpha and 6-keto-PGF1 alpha in 6-day and 10-day corpora lutea, but not in 4-day or 18-day corpora lutea. In the 6- and 10-day corpora lutea, progesterone treatment resulted in a greater inhibition of PGF2 alpha than 6-keto-PGF1 alpha production. Therefore, progesterone treatment brought about an increase in the 6-keto-PGF1 alpha to PGF2 alpha ratio in these cells (12.9 vs. 21.3). It is concluded from these studies that progesterone can modulate luteal prostacyclin and PGF2 alpha synthesis, suggesting an interaction of progesterone and prostaglandin production within the corpus luteum.

Intercellular communication in the bovine corpus luteum

There is a growing body of evidence that intercellular communication is important in the regulation of luteal function. Although the nature of the interactions between small and large luteal cells are not yet clear, it seems likely that they do exist. Many of the substances to which luteal cells respond, such as prostaglandins, growth factors, oxytocin and progesterone, are produced locally. These substances may act as paracrine factors to modulate the response of luteal cells to hormonal signals. Endothelial cells also produce factors that can modify steroidogenesis, and luteal cell-stimulation of endothelial cell proliferation is necessary for the extensive angiogenesis that occurs during luteinization Finally, bidirectional intercellular communication likely occurs between luteal cells and resident immune cells. Immune cells produce cytokines that can modify progesterone and prostaglandin synthesis by luteal cells. Cytokines may also have direct cytotoxic effects on luteal cells, and dead cells are then phagocytized by resident macrophages. Also, factors secreted by luteal cells can serve as chemoattractants for immune cells, and can enhance or suppress immune cell functions. There is little doubt that intercellular communication within the corpus luteum is very complex. One must remember, however, that nearly all evidence collected thus far is based on in vitro studies. Eventually, technology will allow for study of these interactions in vivo, and may lead to new methods for control of luteal function.

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.

Progesterone regulation of luteinizing hormone receptors on cultured bovine luteal cells.

During development of the corpus luteum (CL), the numbers of luteinizing hormone (LH) receptors increase. Cultured bovine luteal cells from developing and mature CL were used to examine the influence of progesterone (P4) on this receptor. CL were obtained from dairy cows during the early or middle phase of the estrous cycle. In early CL, the number of receptors per cell was increased by exogenous progesterone treatment but there was no effect on receptor numbers in cells from midcycle CL. Binding affinities did not change with respect to age or treatment. Forskolin elevated endogenous progesterone and also enlarged the receptor population. The action did not appear to be an unmasking of cryptic receptors since the effect was not seen in luteal particulates. Elevation of LH receptor numbers by progesterone in immature CL may be a form of intraluteal regulation contributing to the functional maturation of these steroidogenic cells.

The Interface of the Immune and Reproductive Systems in the Ovary: Lessons Learned from the Corpus Luteum of Domestic Animal Models

Citation Pate JL, Toyokawa K, Walusimbi S, Brzezicka E. The interface of the immune and reproductive systems in the ovary: lessons learned from the corpus luteum of domestic animal models. Am J Reprod Immunol 2010

Mechanism of action of TNF-α-stimulated prostaglandin production in cultured bovine luteal cells

Abstract Tumor necrosis factor-alpha (TNF-α) influences hormone synthesis of many ovarian cell types and can also exert cytotoxic effects, possibly by increasing the synthesis of prostaglandins. The purpose of the present study was to characterize the mechanism of TNF-α-stimulated prostaglandin F2α (PGF2α) production in cultured bovine luteal cells. Inhibitors of RNA and protein synthesis (actinomycin D and cycloheximide, respectively) completely blocked TNF-α-stimulated PGF2α production. The phospholipase A2 inhibitor, aristolochic acid, prevented TNF-α-stimulated, but not basal, PGF2α production, whereas the phospholipase C inhibitor, compound 48 80 , was without effect. The addition of arachidonic acid to cultures did not overcome the inhibitory effects of cycloheximide or aristolochic acid. In conclusion, TNF-α-stimulated prostaglandin production by bovine luteal cells is dependent upon the stimulation of phospholipase A2 through mechanisms which require synthesis of RNA and protein.

PHYSIOLOGY AND ENDOCRINOLOGY SYMPOSIUM: Role of immune cells in the corpus luteum

The immune system is essential for optimal function of the reproductive system. The corpus luteum (CL) is an endocrine organ that secretes progesterone, which is responsible for regulating the length of the estrous cycle, and for the establishment and maintenance of pregnancy in mammals. This paper reviews literature that addresses 2 areas; i) how immune cells are recruited to the CL, and ii) how immune cells communicate with luteal cells to affect the formation, development, and regression of the CL. Immune cells, primarily recruited to the ovulatory follicle from lymphoid organs after the LH surge, facilitate ovulation and populate the developing CL. During the luteal phase, changes in the population of macrophages, eosinophils, neutrophils, and T lymphocytes occur at critical functional stages of the CL. In addition to their role in facilitating ovulation, immune cells may have an important role in luteal function. Evidence shows that cytokines secreted by immune cells modulate both luteotropic and luteolytic processes. However, the decision to pursue either function may depend on the environment provided by luteal cells. It is suggested that understanding the role immune cells play could lead to identification of new strategies to improve fertility in dairy cattle and other species.

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.