Identification of Novel Genes Regulated by the Luteinizing Hormone in the Primate Corpus Luteum: Insight into Their Regulation During the Late Luteal Phase

The process of luteinization, during which granulosa cells are transformed into luteal cells, is accompanied by dramatic changes in the response of luteal cells to LH. Although luteal cells require LH-cAMP signalling cascade for survival, whether these cells respond to trophic factors through changes in gene expression remain poorly characterized. In an attempt to characterize gonadotropin (LH)-regulated gene expression in the bonnet monkey corpus luteum (CL), changes in gene expression after GnRH antagonist treatment to inhibit LH secretion, different stages of CL and during hCG-simulated early pregnancy were examined using DDRT-PCR, Northern blot and semiquantitative RT-PCR analyses. We have identified 7 non-redundant cDNAs whose expression were regulated by LH. The results show that inhibition of LH secretion not only leads to down-regulation in the expression of genes viz., LDL receptor and Aldose reductase, but expression of some of the genes was up-regulated viz., Humanin, RNA helicase, Lyric protein, Acidic ribosomal phosphoprotein and KIAA1750. Messenger RNA levels of the genes identified as up-regulated after LH inhibition were higher during late- compared to the early- and mid-luteal phase CL, but treatment with hCG down-regulated their expressions. We conclude that we have identified novel genes (known and unknown) that are up-or down-regulated by LH, and the results suggest that LH-mediated activation and repression of expression of many genes is central to the regulation of structure and function of CL in the monkey

Determination of onset of apoptosis in granulosa cells of the preovulatory follicles in the bonnet monkey (Macaca radiata): correlation with mitogen-activated protein kinase activities

During reproductive life, only a selected few ovarian follicles mature and ovulate, while the vast majority of follicles undergo a degenerative process called atresia. Recent studies have indicated that follicular atresia is mediated through apoptosis of follicular granulosa cells. The objectives of the present study were to determine the time of onset of apoptosis in granulosa cells of preovulatory follicles and to evaluate the consequences of gonadotropin withdrawal on mitogen-activated protein (MAP) kinase activities. Bonnet monkeys (Macaca radiata) undergoing controlled ovarian stimulation cycles were utilized for stimulation of multiple follicles, and granulosa cells were retrieved from preovulatory follicles at 24, 48, 72, and 96 h after stopping gonadotropin treatment. Serum and follicular fluid estradiol concentrations were highest at 24 h but declined precipitously (P < 0.05) to reach the lowest concentrations at 96 h; however, progesterone concentrations during this period did not increase, indicating the absence of luteinization. Quantitative analysis of genomic DNA by 3'-end labeling revealed the presence of low-molecular-weight fragments from 48 h onward, but by agarose gel electrophoresis, DNA laddering could be visualized only after 72 h. Messenger RNA expression for Bax, caspase-2, and caspase-3 increased with the onset of apoptosis. Immunoblot analysis of MAP kinases in lysates of granulosa cells (48-72 h) indicated increased (P < 0.05) levels of phosphorylated extracellular response kinase-1 and -2, Jun N-terminal kinase (JNK)-1 and -2, and p38. However, in vitro kinase assay data indicated that only phospho-JNK and -p38 activities were higher at 72 h compared to 24 h. These results demonstrate that granulosa cells of preovulatory follicles undergo apoptosis and that increased activities of phospho-JNK and -p38 are correlated with apoptosis in the primate

Cloning of a buffalo (Bubalus bubalis) prostaglandin F_2_\alpha receptor: changes in its expression and concentration in the buffalo cow corpus luteum

Acting primarily through its specific G protein-coupled receptor termed FPr, prostaglandin (PG) F_2_\alpha) induces regression of the corpus luteum (CL) at the end of a non-fertile oestrous cycle. This study was aimed at cloning a full-length cDNA for FPr and determining its expression and protein concentrations during different stages of CL development in the water buffalo. Serum progesterone and StAR expression were determined to establish temporal relationships between indices of steroidogenesis and changes in FPr expression at different stages of CL development. In contrast to the dairy cow, the stage IV CL (day 20 of the oestrous cycle) did not appear to be functionally regressed in the buffalo. Molecular cloning of a cDNA encoding the buffalo FPr yielded a full length 2193 bp FPr cDNA containing a single open reading frame encoding a 362 amino acid protein with seven putative membrane-spanning domains. The deduced buffalo FPr amino acid sequence possesses a high degree of identity with the other mammalian homologues. Steady state concentration of buffalo FPr transcript increased (P > 0.05) from stage I to stage II/III, and declined at 18 h post PGF_2_\alpha injection. The FPr concentration expressed as fmol/microg of plasma membrane protein showed an increase (P > 0.05) from stage I (1.98 \pm 0.10), through stage II/III (2.42 \pm 0.48) to stage IV (2.77 \pm 0.18). High affinity FPr was observed in stage I (K(d) 4.86 nmol) and stage II/III ($K_d$ 6.28 nmol) while low affinity FPr ($K_d$ 19.44 nmol) was observed in stage IV. In conclusion, we have cloned a full length FPr cDNA from buffalo cow CL and observed that FPr mRNA expression, receptor number and affinity did not vary significantly (P > 0.05) within the luteal phase of the oestrous cycle

Peripheral priming induces plastic transcriptomic and proteomic responses in circulating neutrophils required for pathogen containment

Neutrophils rapidly respond to inflammation and infection, but to which degree their functional trajectories after mobilization from the bone marrow are shaped within the circulation remains vague. Experimental limitations have so far hampered neutrophil research in human disease. Here, using innovative fixation and single-cell-based toolsets, we profile human and murine neutrophil transcriptomes and proteomes during steady state and bacterial infection. We find that peripheral priming of circulating neutrophils leads to dynamic shifts dominated by conserved up-regulation of antimicrobial genes across neutrophil substates, facilitating pathogen containment. We show the TLR4/NF-κB signaling-dependent up-regulation of canonical neutrophil activation markers like CD177/NB-1 during acute inflammation, resulting in functional shifts in vivo. Blocking de novo RNA synthesis in circulating neutrophils abrogates these plastic shifts and prevents the adaptation of antibacterial neutrophil programs by up-regulation of distinct effector molecules upon infection. These data underline transcriptional plasticity as a relevant mechanism of functional neutrophil reprogramming during acute infection to foster bacterial containment within the circulation