Indian Journal of Animal Research

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Indian Journal of Animal Research, volume 57 issue 6 (june 2023) : 702-707

Effect of Tumor Necrosis Factor-α on in vitro Prostaglandin Production in Buffalo Corpus Luteum

M.K. Tripathi2,*, S. Mondal1, I.J. Reddy1, A. Mor1
1ICAR-National Institute of Animal Nutrition and Physiology, Bengaluru-560 030, Karnataka, India.
2Division of Livestock and Fishery Management, ICAR-Research Complex for Eastern Region, Patna-800 014, Bihar, India.
Cite article:- Tripathi M.K., Mondal S., Reddy I.J., Mor A. (2023). Effect of Tumor Necrosis Factor-α on in vitro Prostaglandin Production in Buffalo Corpus Luteum . Indian Journal of Animal Research. 57(6): 702-707. doi: 10.18805/IJAR.B-4346.
Background: Corpus luteum plays key role in embryonic survival. Prostaglandins are the important regulator controlling the life span of corpus luteum. The present study investigated the effect of various doses of TNFα on in vitro PGF and PGE2 production and expression profiling of PGFS and PGES mRNA in buffalo Corpus Luteum (CL).

Methods: Buffalo ovaries with mid-luteal phase CL were collected from the abattoir and CL were enucleated from surrounding tissues. Corpus luteum were finely chopped, rinsed with HBSS (Hanks Balanced Salt Solution) medium; supplemented with gentamycin and 0.1% BSA and incubated at 37°C for 1 hr in HBSS containing 0.1% collagenase. The cell suspension following filtration was washed by HBBS supplemented with gentamycin and 0.1% BSA (bovine serum albumin) and was treated with increasing doses of TNFα (0.1, 0.5 and 1.0 nM) and cultured at 38.5°C, 5% CO2 level for 24 hr. 

Result: There was dose dependent increase in concentrations of PGF and PGE2 with increasing doses of TNFα. The PGFS (prostaglandin F synthase) mRNA expression increased with increasing doses of TNFα. However, there was decrease in PGES (prostaglandin E synthase) mRNA expression at 0.1 nM and 0.5 nM TNFα but PGES mRNA expression increased at 1.0 nM TNFα as compared to control. It can be concluded that TNFα may alter PGES and PGFS mRNA expression and prostaglandin secretion in buffalo CL. 
Premature luteal regression or inadequate luteal function is a major cause of early embryonic mortality in domestic ruminants including buffaloes. Early embryonic mortality is a major cause of reproductive failure in buffaloes during pre-implantation period (Mondal and Prakash 2002; Mondal et al., 2009; Mondal et al., 2010) and accounts for 20% loss in buffaloes living closer to equator (Vale et al., 1989). Prostaglandins namely prostaglandin E2 (PGEα) and prostaglandin F2a (PGF) play important role during maternal recognition of pregnancy, implantation and establishment of pregnancy in livestock including buffalo (Nandi et al., 2012; Mondal, 2013). The life span of CL is controlled by complex interactions among various luteotrophic and luteolytic factors including prostaglandins. Prostaglandin E2 protects the CL from spontaneous regression and play important role in maintenance of pregnancy (Akinlosotu et al., 1986; Christenson et al., 1994) whereas PGF primarily secreted from the surface epithelium of the uterus cause regression of CL (Davis and Rueda, 2002). In spite of PGF produced from endometrium, its secretion is also reported from CL in cows (Townson and Pate, 1994, 1996) and pigs (Diaz et al., 2000). The small amounts of uterine PGF stimulate the production of PGF in the CL which indicates the presence of a positive feedback pathway in the ruminant CL (Diaz et al., 2002).  PGF can autoregulate its synthesis in CL and it has been proposed that PGF produced locally in CL acts via a paracrine and/or autocrine mechanism to induce luteolysis (Wiltbank and Ottobre, 2003). Progesterone, oxytocin and various cytokines have been shown to affect luteal PGF production in different species (Pate, 1988; Wiltbank and Ottobre, 2003, Tripathi et al. 2018, Grazul et al., 1989).

Tumour Necrosis Factor-alpha (TNFα), a cytokine possessing wide biological actions, is mainly produced by ovarian macrophages and increased during regression of the CL (Penny et al. 1999). TNFα participates in the regression of the corpus luteum in bovine (Pate, 1995). Highest expression of its specific receptors (TNF-RI) is present in the bovine corpus luteum during luteolysis (Friedman et al. 2000; Korzekwa et al. 2008). The luteolytic action of TNFα may be mediated by the intraluteal production of PGF2a as TNFa dramatically stimulated PGF production by cultured luteal cells of cow (Benyo and Pate, 1992).  There is an integrated role of luteal PGF and PGE2 in auto-regulation of CL function (Arosh et al., 2004). Intrauterine administration of PGEhad been found to protect the CL from spontaneous and/or induced luteolysis in ruminants (Pratt et al., 1977). The production of endometrial prostaglandins is governed by the enzymes prostaglandin E and F synthase (PGES and PGFS) which convert PGH2 to PGE2 and PGF, respectively. There is paucity of information regarding the role of TNFa in regulation of PGF2a and PGE2 secretion and expression of PGES and PGFS mRNA in buffalo CL. Keeping this in view the present work was undertaken to investigate the effect of TNFa on in vitro prostaglandin production and expression of PGES and PGFS mRNA in buffalo CL.
Collection of corpus luteum and identification of stage of estrous cycle
Buffalo ovaries with CL were collected from a local abattoir within 1 hr after exsanguinations. The ovaries were then submerged in ice-cold physiological saline (0.9% NaCl; pH 7.4) containing antibiotics [penicillin (10 IU/ml), streptomycin (100 g/ml), amphotericin (2 g/ml) and L-glutamine (100 μg/ml)] and transported to the laboratory. The stages of the estrous cycle were determined according to previously described methods (Mondal et al., 2004; Ghosh and Mondal, 2006, Chouhan et al., 2013) and were categorized to Stage I (days 3 to 5 of the cycle), Stage II (days 6 to 15 of the cycle) and Stage III (days 16 to 21 of the cycle). The Stage I is the developing stage, Stage II is mature one while Stage III is in the regressed stage of corpus luteum. The mid-luteal phase of estrous cycle was determined by macroscopic examination of the CL by observing the change of colour from dark red to orange, firm in consistency on palpation and demarcated from rest of the ovary (Mondal et al., 2013).
Luteal cell Isolation
Mid stage corpus luteum were immediately separated from the surrounding tissues of the ovaries, chopped, rinsed a few times with Hanks Balanced Salt Solution (HBSS, H9394 Sigma) medium supplemented with gentamycin (50 μg/μl, pH 7.4, TC026, Hiimedia) and 0.1% bovine serum albumin (BSA, RM3158, Himedia) and incubated at 37°C for 1 hr in HBSS containing 0.1% collagenase (Type II collagenase from Clostridium histolyticum, pH 7.4, C6885, Sigma). The cell suspension obtained from the digestion was filtered through plastic strainer (70 μM) to remove undissociated tissue fragments. There after the filtrate was washed three times by centrifugation at 600 x g for 10 min with HBBS supplemented with gentamycin and 0.1% BSA. The number of viable cells that excluded Trypan blue was counted using a haemocytometer.   
Culture of luteal cells and treatment with TNFa
After cell counting and viability determination the luteal cells were seeded at the rate of 1×105 viable cells in RPMI (Roswell Park Memorial Institute) 1640 medium supplemented with 100µg glutamine, 1µl gentamicin (50 µg/µl; TC026-1G, Himedia) and 10% FBS (Fetal Bovine Serum; F2442, Sigma) and were treated with increasing doses of TNFa (0.1, 0.5 and 1.0 nM) followed by incubation at 38.5°C and 5% CO2 level for 24 hours. Cells along with spent media were harvested and centrifuged at 8000 rpm for 6 minutes to isolate cell pellet. The spent media was collected for quantification of prostaglandins. The cell pellet was kept in 100 µl RLT-lysis buffer (Qiagen, Germany) containing β-mercaptoethanol and stored at -80°C for expression of PGEs and PGFS mRNA.
Quantification of prostaglandins
PGF2a concentrations were estimated in 50 ml aliquots of culture medium after 10fold dilution with extraction buffer using ELISA kit (404710, Neogen, USA). The sensitivity of the assay was 0.002 ng/ml. The cross reactivity of the antisera against 6-keto prostaglandin F1a, 13, 14 dihydro-15 keto-prostaglandin F2a, prostaglandin D2 and prostaglandin E2 were 3.05%, 0.05%, 0.05% and <0.01%, respectively. The intra- and inter-assay coefficients of variation were less than 15%. Concentrations of PGE2 were estimated in 50 ml aliquots of culture medium after 5fold dilution with extraction buffer. The sensitivity of the assay was 0.002 ng/ml. The cross reactivity of the antisera against 6-keto prostaglandin F1, 13,14 dihydro-15 keto-prostaglandin F2a, prostaglandin D2 and prostaglandin F2a were >0.01% for all. Intra and inter-assay coefficients of variation were less than 13%.
RNA isolation
Cultured cell pellets of both the treatment and control groups were used for the extraction of total RNA using RNeasy Minikit (M/s Qiagen, Germany) as per manufacturer’s  protocol and quantified by nanodrop spectrophotometer (Eppendorf, Germany) at 260 nm and 280 nm. RNA integrity was checked by formaldehyde-agarose gel (1%) electrophoresis and visualized under UV light after staining with ethidium bromide.
Reverse transcription polymerase chain reaction (RT-PCR)
cDNA synthesis kit (Biorad iScriptTM cDNA synthesis kit, 1708890, Biorad, USA) was used to synthesize cDNA following the protocol - I script reverse transcriptase (1μl), 5X iScript reaction mix (4µl), Nuclease free water (14 µl) and (100 ng) RNA (1 µl) to make total volume 20 µl. The reactants were  incubated at 25°C for 5 min, 42°C for 30 min followed by 85°C for 5 min. Following first strand synthesis, PCR amplification of PGES and PGFS were carried out using gene specific primers designed on the basis of aligned nucleotide sequences available in GenBank (Table 1). PCR amplification was carried out in 25 µl total volume. Reaction mixture contained of 2 µl first strand of cDNA, 10X Buffer (2.5 µl), 50 mM dNTP Mix (0.5 µl), 25 mM MgCl2 (2.5µl), forward primer (1 µl), reverse primer (1 µl), Mili Q water (15.3 µl) and 0.2 µl Taq DNA Polymerase (5U/µl). Following initial denaturation at 95°C for 5 min, amplification (30 cycles) was carried out under the conditions mentioned in Table 1, followed by final extension at 72°C for 10 min. The generated cDNA fragments were resolved by agarose gel (1%) electrophoresis.

Table 1: Gene specific primers used for amplification of PGES and PGFS cDNA.

Real time PCR
PCR analyses were performed with three replicates per sample of each gene using the STEP ONE PLUS real time PCR system (Applied Bio systems, USA), Fast SYBR Green Master Mix (KK4604, Applied Bio systems, USA) and gene specific primers for both housekeeping and target genes (Table 1). Primer 3 program (Rozen and Skaletsky, 2000) was used for designing the primers with an annealing temperature of 59°C and amplification size of less than 250 bp. Primer efficiencies were checked by 10fold serial dilution of cDNAs (Svec et al., 2015) and ranged between 95 and 105%. β-actin was used as endogenous control. Thermal cycling was carried out as per manufacturer’s protocol (95°C, 20 sec followed by 40 cycles of 95°C, 3 sec and 60°C, 25 sec and a melt curve of 95°C (15 sec), 60°C (1 min) and 95°C (15 sec). The specificity of each PCR product was determined by melt curve analysis and amplicon size determination was carried out by agarose gel electrophoresis (2%). Negative control consisted of all the components of qRT-PCR mix except cDNA was used for all primers. The relative quantification of gene expression changes were recorded after normalizing for β-actin expression computed by using 2DDCT method (Svec et al., 2015) in which CT value from controls serves as calibrator.
Statistical analysis
The results are shown as the Mean±S.E.M. of values obtained from separate experiments, each performed in triplicate. The differences in concentrations of PGF2a, PGE2 and transcript abundance were analyzed by ANOVA followed by Tukey’s multiple comparison tests using the statistical package of Graph Pad Prism 5, San Diego, USA.  A value of p<0.05 was considered statistically significant.
The effect of various doses of TNFa (0.1 nM, 0.5 nM, 1.0 nM) on in vitro PGF2a  production in buffalo luteal cells is presented in Fig 1(a). The concentrations of PGF2a. were 110.2±11.53, 122.6±19.04, 124.6±22.68, 130.4±15.96 pg/ml in control, 0.1 nM, 0.5 nM and 1.0 nM TNFα treatment group, respectively. The effect of various doses of TNFα (0.1 nM, 0.5 nM and 1.0 nM) on in vitro PGEproduction in buffalo luteal cells is presented in Fig 1 (b). The levels of PGE2 were 2.27±0.36, 2.84±0.37, 2.99±0.42 and 3.17±0.57 pg/ml in control, 0.1 nM, 0.5 nM and 1.0 nM TNFa, respectively.

Fig 1: Effect of TNFá on PGF2á and PGE2 production by luteal cells in buffalo.

The effect of different doses (0.1 nM, 0.5 nM, 1.0 nM) of TNFa on PGES and PGFS mRNA expression is presented in Fig 2(a) and 2(b). TNFa causes dose dependent increase in PGFS mRNA expression. However, there was no significant difference in PGFS expression among different groups. There was non-significant (P>0.05) decrease in PGES mRNA expression at 0.1 nM and 0.5 nM but increase at 1.0 nM concentrations of TNFa in comparison to control group. However, there was no significant differences (P>0.05) in PGES and PGFS mRNA expression among various groups.

Fig 2: Effect of increasing concentrations of TNFa on PGES (a) and PGFS mRNA expression in buffalo luteal cell.

To the best of our knowledge and belief, this is the first study to report the effect of TNFα on in vitro prostaglandin production and expression of PGFS and PGES mRNA in buffalo Corpus luteum. The present study showed that TNFα. stimulated PGF and PGE2 production by cultured buffalo luteal cells. There was dose dependent increase in the concentrations of both PGF and PGE2 with the increasing doses of TNFα. However, no significant difference in PGF and PGE2 concentrations were found among the different treatment groups. Since early corpus luteum is relatively resistant to PGF, mid stage CL was chosen in the present study to see the effect of TNFα on luteal cell PGF production. Our results are in alignment with the earlier report of Benyo and Pate (1992) wherein TNFa stimulated PGF secretion by the cells in a dose-dependent fashion in mid-stage bovine CL. Similarly, Sakumoto et al., (2000a) reported that TNFa significantly stimulated both PGF and PGE2 secretion in the bovine luteal cells in a dose-dependent fashion. In porcine species also TNFa had been shown to stimulate both PGF and PGE2 secretion in a dose-dependent manner in cultured mid luteal cells (Miyamoto et al., 2002). The production of both PGE2 and PGF2a was dose-dependently increased by recombinant human TNFα (rTNFα) in cultured human granulosa-lutein cells (Wang et al. 1992). However, our results are not in agreement with the findings of Skarzynski et al., (2003) and Korzekwa et al., (2008) who reported that infusion of lower concentration of TNFa caused the increase in plasma PGF2a level and luteolysis whereas its higher concentration stimulated PGE2 level and prolongs the estrous cycle  in cattle. (Skarzynski et al., 2003, Korzekwa et al., 2008) which may be due to species difference.  The stimulatory effect of TNFα on PGF2α/PGE2 had also been shown in various tissues of different species including bovine endometrium (Miyamoto et al., 2000; Okuda et al., 2002; Skarzynski et al., 2003), equine endometrium (Szóstek et al. 2014), porcine endometrium (Blitek and Ziecik, 2006; Waclawik et al., 2010), porcine maternal placenta (Jana et al., 2008), human luteal phase endometrial cells (Chen et al., 1995) and human fetal membranes (Kent et al., 1993).

Most prostaglandins are produced from arachidonic acid (AA) which is released from the membrane phospholipids via the action of Cytosolic phospholipase A2 (cPLA2) (Wiltbank and Ottobre, 2003). Cyclooxygenases (COX-1 and COX-2) convert AA into PGH2, the common metabolite for various PGs including PGE2 and PGF. PGE synthase (PGES) and PGF synthase (PGFS), catalyze the formation of PGE2 and PGF respectively from PGH2 (Smith and Dewitt, 1996). The bovine CL possesses all of the PGE2 and PGF machineries necessary for autoregulation of its function and PGE2 and PGF machineries are selectively and specifically expressed in CL (Arosh et al., 2004). The present study showed that TNFa increased PGFS mRNA expression in dose dependent manner. Our findings gain support from the finding of Korzekwa et al., (2011) who reported that TNFα increased mRNA expression of PGFS in immortalized bovine luteal endothelial cell line. In our results the stimulatory effect of TNFα on both PGF and PGEproduction indicated that the regulation of synthesis of both PGs by TNFa may be at some common sites like substrate availability, phospholipase A2 or cyclooxygenase enzyme activity as well at PGFS level in case of PGF synthesis but it may not be at PGES level in case of PGE2 synthesis as Parent et al., (2002) reported that TNFa stimulated PGE2 output but not COX-2 expression. Therefore, it is possible that TNFα may stimulate the production of PGE2 by increasing the availability of arachidonic acid which is the common substrate for PGs synthesis. Another source of PGF is PGE2, which could be converted to PGF by PGE2 9-keto reductase (Duffy, 2015).  TNFa stimulated PGF secretion through stimulation of phospholipase A2 (PLA2) and MAPK pathways in bovine luteal cells (Sakumoto et al., 2000b). TNFa is potent stimulators of luteal prostaglandin production and appears to act primarily by stimulating phospholipase A2, (Pate, 1995). TNFα stimulated PGF production by luteal cells is dependent upon the stimulation of phospholipase A2 through mechanisms requiring synthesis of RNA and protein (Townson and Pate 1996). The mechanism of action of TNFa on PGE2 is not well documented in luteal cell. However, Waclawik et al., (2010) reported that TNFa significantly stimulated PGE2 synthesis and release through up-regulation of prostaglandin-endoperoxide synthase 2 mRNA, as well as PGEsynthase (mPGES1) mRNA and protein expression in endometrial luminal epithelial cells of porcine collected from days 11-12 of the estrous cycle and pregnancy. Similarly, in another experiment in equine endometrium, Szóstek et al. (2014) reported that TNFα stimulated PGE2 production to a greater extent and PGF2α secretion to a lower level through up-regulation of PG synthases mRNA transcription. 
The overall result indicates that TNFα may serve as an important modulator of prostaglandins production in the developing stage of buffalo corpus luteum; however, the possible physiological role of TNFα for simultaneous increase of intra-luteal PGF and PGE2 production seems counterintuitive and needs further detailed studies to clarify its precise role.
We thank Director, NIANP for providing the necessary facilities for conducting the research work. Thanks are due to Director, CIRB for granting permission to conduct research work at NIANP Bangalore, India.

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