Ryosuke Sakumoto

Tumor Necrosis Factor-α and Its Receptor in Bovine Corpus Luteum Throughout the Estrous Cycle

Abstract The objective of this study was to investigate tumor necrosis factor α (TNF-α) expression, the presence of functional TNF-α receptors, and expression of TNF receptor type I (TNF-RI) mRNA in the bovine corpus luteum (CL) during different stages of the estrous cycle. Reverse transcription (RT)-polymerase chain reaction (PCR) showed no difference in TNF-α mRNA expression during the estrous cycle. Concentrations of TNF-α in the CL tissue increased significantly from the mid to the late luteal stage and decreased thereafter (P < 0.05). An RT-PCR analysis showed higher levels of TNF-RI mRNA in CL of Days 3–7 than of other stages (P < 0.05). 125I-TNF-α binding to the membranes of bovine CL was maximal after incubation at 38°C for 48 h. The binding was much greater for TNF-α than for related peptides. A Scatchard analysis revealed the presence of a high-affinity binding site in the CL membranes collected at each phase of the estrous cycle (dissociation constant: 3.60 ± 0.58–5.79 ± 0.19 nM). In contrast to TNF-RI mRNA expression, the levels of receptor protein were similar at each stage of the estrous cycle. When cultured cells of all luteal stages were exposed to TNF-α (1–100 ng/ml), TNF-α stimulated prostaglandin F2α and prostaglandin E2 secretion by the cells in a dose-dependent fashion (P < 0.01), especially during the early luteal phase, although it did not affect progesterone secretion. These results indicate the local production of TNF-α and the presence of functional TNF-RI in bovine CL throughout the estrous cycle, and suggest that TNF-α plays some roles in regulating bovine CL function throughout the estrous cycle.

Multiple roles of TNF super family members in corpus luteum function

The main function of the corpus luteum (CL) is the production of progesterone. Adequate luteal progesterone is crucial for determining the physiological duration of the estrous cycle and for achieving a successful pregnancy. The CL is regulated not only by hypophyseal gonadotropin, but also by a number of cytokines that are locally produced. Tumor necrosis factor-α (TNF) and its specific receptors (TNFR) are present in the CL of many species. TNF plays multiple and likely important roles in CL function throughout the estrous cycle. TNF appears to have luteotropic and luteolytic roles in the CLs. In contrast, Fas ligand (Fas L), another member of TNF super family (TNF-SF), is primarily recognized for its apoptotic actions. Presumably, Fas L binds its cognate receptor (Fas) to induce structural luteolysis. This review is designed to focus on recent studies documenting the expression of TNF and Fas L, their receptors, and intracellular signaling mechanisms in the CL.

Cortisol Is a Suppressor of Apoptosis in Bovine Corpus Luteum

Glucocorticoid (GC) acts as a modulator of physiological functions in several organs. In the present study, we examined whether GC suppresses luteolysis in bovine corpus luteum (CL). Cortisol (an active GC) reduced the mRNA expression of caspase 8 (CASP8) and caspase 3 (CASP3) and reduced the enzymatic activity of CASP3 and cell death induced by tumor necrosis factor (TNF) and interferon gamma (IFNG) in cultured bovine luteal cells. mRNAs and proteins of GC receptor (NR3C1), 11beta-hydroxysteroid dehydrogenase type 1 (HSD11B1), and HSD11B2 were expressed in CL throughout the estrous cycle. Moreover, the protein expression and the enzymatic activity of HSD11B1 were high at the early and the midluteal stages compared to the regressed luteal stage. These results suggest that cortisol suppresses TNF-IFNG-induced apoptosis in vitro by reducing apoptosis signals via CASP8 and CASP3 in bovine CL and that the local increase in cortisol production resulting from increased HSD11B1 at the early and midluteal stages helps to maintain CL function by suppressing apoptosis of luteal cells.

Tumor Necrosis Factor-α (TNF α) Inhibits Progesterone and Estradiol-17β Production from Cultured Granulosa Cells: Presence of TNFα Receptors in Bovine Granulosa and Theca Cells

The aim of this study was to investigate whether functional tumor necrosis factor-α (TNFα) receptors are present in the granulosa cells and the cells of theca interna (theca cells), obtained from bovine follicles classified into one of three groups. Each group was defined as either small vesicular ovarian follicles (small follicles; 3-5 mm in diameter), preovulatory mature ovarian follicles (preovulatory follicles) or atretic follicles (12-18 mm) according to gross examination of the corpus luteum in the epsilateral or contralateral ovary and the uterus (size, color, consistency and mucus), and the ratio of progesterone (P4) and estradiol-17β (E2) concentrations in follicular fluid. A Scatchard analysis showed the presence of a high-affinity binding site on both granulosa and theca cells from all follicles examined (dissociation constant: 4.7 ± 0.15 to 6.9 ± 1.40 nM). Moreover, TNFα receptor concentrations in granulosa and theca cells obtained from atretic follicles were significantly higher than those in the cells from preovulatory follicles (P<0.05). Exposure of cultured granulosa cells from small antral follicles to recombinant human TNFα (rhTNFα; 0.06-6 nM) inhibited E2 secretion in a dose-dependent fashion (P<0.01), but did not affect P4 secretion. In addition, rhTNFα inhibited follicle stimulating hormone-, forskolin- or dibutylyl cyclic AMP-induced P4 and E2 secretion by the cells (P<0.01). These results indicate the presence of functional TNFα receptors in bovine granulosa and theca cells in small, preovulatory and atretic follicles, and suggest that TNFα plays a role in regulating their secretory function.

Localization of gene and protein expressions of tumor necrosis factor-α and tumor necrosis factor receptor types I and II in the bovine corpus luteum during the estrous cycle

One of the many roles of tumor necrosis factor (TNF)-α is to control mammalian corpus luteum (CL) PG synthesis and apoptotic cell death. Here, the cellular localization of TNF-α and its type I (TNF-RI) and type II (TNF-RII) receptors in bovine luteal tissue were analyzed using in situ hybridization, immunohistochemistry, and quantitative real-time PCR. Transcripts for TNF-α were expressed in bovine CL throughout the estrous cycle, but were significantly more abundant (P < 0.01) at the regressed luteal stage than at the other stages. Localization of TNF-α transcripts and protein were observed in large and small bovine luteal cells, as well as in immune cells. Moreover, transcripts for TNF-RI and TNF-RII were expressed in bovine CL throughout the estrous cycle. The abundance of TNF-RII transcripts was greater (P < 0.01) at the regressed luteal stage than at the other stages, whereas TNF-RI transcript abundance did not significantly change. Expression of TNF-RI and TNF-RII transcripts and proteins were observed in both the large and small luteal cells, and the proteins were also expressed in the immune cells and vascular endothelial cells. These results suggest that TNF-α sources include immune cells, as well as large and small luteal cells, and that TNF-RI and TNF-RII are present in the luteal cells of the bovine CL.

Anti-Apoptotic Roles of Prostaglandin E2 and F2alpha in Bovine Luteal Steroidogenic Cells

Abstract Production of prostaglandins (PGs) and expression of their receptors have been demonstrated in bovine corpus luteum (CL). The aim of the present study was to determine whether PGE2 and PGF2alpha have roles in bovine luteal steroidogenic cell (LSC) apoptosis. Cultured bovine LSCs obtained at the midluteal stage (Days 8–12 of the cycle) were treated for 24 h with PGE2 (0.001–1 μM) and PGF2alpha (0.001–1 μM). Prostaglandin E2 (1 μM) and PGF2alpha (1 μM) significantly stimulated progesterone (P4) production and reduced the levels of cell death in the cells cultured with or without tumor necrosis factor alpha (TNF)/interferon gamma (IFNG), in the presence and absence of FAS ligand (P < 0.05). Furthermore, DNA fragmentation induced by TNF/IFNG was observed to be suppressed by PGE2 and PGF2alpha. Prostaglandin E2 and PGF2alpha also attenuated mRNA expression of caspase 3 and caspase 8, as well as caspase 3 activity (P < 0.05) in TNF/IFNG-treated cells. FAS mRNA and protein expression were decreased only by PGF2alpha (P < 0.05). A specific P4 receptor antagonist (onapristone) attenuated the apoptosis-inhibitory effects of PGE2 and PGF2alpha in the absence of TNF/IFNG (P < 0.05). A PG synthesis inhibitor (indomethacin) reduced cell viability in PGE2- and PGF2alpha-treated cells (P < 0.05). A specific inhibitor of cyclooxygenase (PTGS), PTGS2 (NS-398), also reduced cell viability, whereas an inhibitor of PTGS1 (FR122047) did not affect it. The overall results suggest that PGE2 and PGF2alpha locally play luteoprotective roles in bovine CL by suppressing apoptosis of LSCs.

Tumor necrosis factor-α and its receptor in the corpus luteum of pregnant cows.

The objective of this study was to investigate the presence of tumor necrosis factor (TNF)‐α mRNA and TNF‐α receptors in the bovine corpus luteum (CL) during the gestation period. TNF‐α mRNA and TNF‐α receptors were determined on bovine CL from pregnant cows at three stages: trimester I (fetal crown‐rump length; 6–20 cm), trimester II (25–45 cm) and trimester III (50–80 cm). TNF‐α mRNA was detected by an RT‐PCR analysis in the CL of all stages of gestation. A Scatchard analysis revealed the presence of a high‐affinity binding site (Kd; 5.1–6.9 nM) in the CL membranes collected at each stage of gestation. Furthermore, the concentrations of TNF‐α receptors in the CL of trimesters I (24.0 ± 1.95 pmol/mg protein) and III (21.6 ± 2.39 pmol/mg protein) of gestation were significantly higher than the concentration in trimester II (14.9 ± 2.07 pmol/mg protein) (P < 0.05). These results indicate that TNF‐α is locally produced and that TNF‐α receptors are present in bovine CL during the gestation period, and suggest that TNF‐α plays one or more roles as a paracrine factor in regulating bovine CL function during the entire gestation period. Mol. Reprod. Dev. 55:406–411, 2000.

Tumor necrosis factor-α stimulates prostaglandin F2α secretion by bovine luteal cells via activation of mitogen-activated protein kinase and phospholipase A2 pathways

It has been well demonstrated that tumor necrosis factor‐α (TNFα) stimulates prostaglandin (PG) F2α secretion by bovine corpus luteum (CL) in vitro. The objective of the present study was to clarify the intracellular signaling pathway of TNFα to stimulate PGF2α production in cultured bovine luteal cells. Bovine luteal cells that were obtained from mid‐ (days 8–12 after ovulation) CL were incubated with TNFα (0.6 nM) and/or various compounds as follows: U‐73122 (an inhibitor of phospholipase [PL] C), ACA (an inhibitor of PL‐A2), H‐89 (an inhibitor of protein kinase [PK] A), calphostin C (an inhibitor of PK‐C), L‐NAME/L‐NORG (inhibitors of nitric oxide synthase), and PD98059 (an inhibitor of mitogen‐activated protein kinase [MAPK] kinase). Although U‐73122 (0.1–10 μM), H‐89 (0.1–10 μM), calphostin C (0.01–1 μM) and L‐NAME/L‐NORG (1–100 μM) did not affect TNFα‐induced PGF2α secretion by the cultured cells, ACA (1–100 μM) and PD98059 (0.1–100 μM) inhibited TNFα‐stimulated PGF2α secretion by the cells in a dose‐dependent fashion (P < 0.05 or lower). These findings suggest that TNFα activates the MAPK and PL‐A2 pathways in bovine luteal cells to stimulate PGF2α secretion. Mol. Reprod. Dev. 56:387–391, 2000.

Proliferation of Luteal Steroidogenic Cells in Cattle

The rapid growth of the corpus luteum (CL) after ovulation is believed to be mainly due to an increase in the size of luteal cells (hypertrophy) rather than an increase in their number. However, the relationship between luteal growth and the proliferation of luteal steroidogenic cells (LSCs) is not fully understood. One goal of the present study was to determine whether LSCs proliferate during CL growth. A second goal was to determine whether luteinizing hormone (LH), which is known have roles in the proliferation and differentiation of follicular cells, also affects the proliferation of LSCs. Ki-67 (a cell proliferation marker) was expressed during the early, developing and mid luteal stages and some Ki-67-positive cells co-expressed HSD3B (a steroidogenic marker). DNA content in LSCs isolated from the developing CL increased much more rapidly (indicating rapid growth) than did DNA content in LSCs isolated from the mid CL. The cell cycle-progressive genes CCND2 (cyclin D2) and CCNE1 (cyclin E1) mRNA were expressed more strongly in the small luteal cells than in the large luteal cells. LH decreased the rate of increase of DNA in LSCs isolated from the mid luteal stage but not in LSCs from the developing stage. LH suppressed CCND2 expression in LSCs from the mid luteal stage but not from the developing luteal stage. Furthermore, LH receptor (LHCGR) mRNA expression was higher at the mid luteal stage than at the developing luteal stage. The overall results suggest that the growth of the bovine CL is due to not only hypertrophy of LSCs but also an increase in their number, and that the proliferative ability of luteal steroidogenic cells decreases between the developing and mid luteal stages.

A novel stereotaxic approach to the hypothalamus for the use of push-pull perfusion cannula in Holstein calves

To determine secretory patterns of growth hormone-releasing hormone (GHRH) and somatostatin (SS) and their roles in the regulation of growth hormone (GH) secretion, a method for collecting hypothalamic perfusates, a push-pull perfusion method was developed in calves. With the use of the stereotaxic apparatus for cattle, a cannula was implanted into the hypothalamus of four male calves based upon cerebral ventriculography. Push-pull perfusates were collected at 10 min intervals for 6h and GHRH and SS concentrations in perfusates and plasma GH concentration were determined by EIAs and RIA, respectively. A cannula was implanted into the hypothalamus based on the image of the third ventricle and maintained for 1 month. GHRH and SS showed pulsatile secretion and the pulses for GHRH and SS were irregular in conscious animals. Neither GHRH nor SS secretion had a clear relationship with GH secretion. In the present study, we thus (1) established a stereotaxic technique for approaching the hypothalamus using cerebral ventriculography for calves, and (2) demonstrated that GHRH and SS secretion were pulsatile but not closely related to GH profile in conscious calves. The technique is useful for the study of the functions of the hypothalamus in the control of pituitary hormones in cattle.