Anomalies in ovary following oral exposure to oxytocin: Mechanistic studies

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Reproductive Toxicology 40 (2013) 24– 34

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Reproductive Toxicology

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Anomalies in ovary following oral exposure to oxytocin: Mechanistic studies

Manjari Mishraa,c, Vivek Mishraa, Bhushan P. Chaudhuria, Vinay K. Khannaa, Sanjay Mehrotrab,Shakir Ali c, Mukul Dasa,∗

a CSIR-Indian Institute of Toxicology Research (IITR), P.O. Box No. 80, Mahatma Gandhi Marg, Lucknow 226001, Indiab Department of Medicine, Chhatrapati Shahuji Maharaj Medical University, Lucknow, Indiac Department of Biochemistry, Jamia Hamdard, New Delhi, India

a r t i c l e i n f o

Article history:Received 10 October 2012Received in revised form 3 April 2013Accepted 8 May 2013Available online xxx

Keywords:OxytocinIEC-6Granulosa cellsOvulationpERK1/2

a b s t r a c t

Ovarian anomalies following oral oxytocin (OT) (1 and 10 ng/100 �l) exposure of female Wistar rat pups(10-day old) for 25 days was undertaken as OT injections are illegally used for milk let down in cattlethereby causing oral exposure to human population from early age. OT exposure resulted in increasedovarian weight, � globulin, total number of follicles, and number of corpus luteum (CLs); indicating higherovulation. The mechanism may involve over-expression of pEGFR followed by downstream pERK1/2 andsubsequently increased ovarian PGE-2 along with enhanced COX-2, HAS-2 & TSG-6 (matrix depositionproteins) and GDF-9 (oocyte factor) proteins, suggesting that oral exposure of OT may affect the physi-ology and function of the ovary. Further, in vitro studies showed increased internalization of OT in IEC-6cells which further supports that orally administered OT may cause altered manifestations as shownabove following internalization in mucosal membrane.

© 2013 Elsevier Inc. All rights reserved.

1. Introduction

Oxytocin (OT), a biological hormone, has a physiological role ofperforming specific functions at different stages of life. For exampleOT stimulates labor by uterine contraction and has been implicatedas a drug during delivery cases [1]. Under normal physiologi-cal conditions in lactating mothers, OT causes contraction of themyoepithelial cells which surround the milk alveoli in the mam-mary gland for milk let-down [2].

In the Indian subcontinent, OT ampules, commercially knownas pitocin or syntocinon, are indiscriminately used to enhance milklet-down following intramuscular injections to cattle. The exoge-nous supplementation of OT is absorbed in blood thereby increasingthe concentration (4- to 5-fold) and is retained for at least 2 h fol-lowing injection to animals [3]. It has been presumed that dueto small size of OT (1 kDa) there is a possibility that it crossesthe blood–milk barrier reaching into the milk and thereby caus-ing changes in the permeability of tight junctions of mammarygland [4]. Our prior study has shown the presence of OT in milksamples [5] which is substantiated by the work of Takeda et al. [6]

Abbreviations: CLs, Corpus leuteum; DMEM, Dulbecco’s Modified Eagle Medium;GCs, Granulosa cells; IEC-6, Intestinal epithelial cells; OT, Oxytocin; OTR, Oxytocinreceptor; PVN, Paraventricular nucleus; SGF, Simulated gastric fluid.

∗ Corresponding author. Tel.: +91 522 2613786; fax: +91 522 2628227.E-mail address: [email protected] (M. Das).

wherein intraperitoneal injection of radiolabelled OT given to ratdams transfer to the plasma of neonates through milk.

A majority of peptides are known to be absorbed with lessbioavailability (0.5–1%) [7]. This may increase according to theconditions found in the gastrointestinal (GI) tract [7–9]. Our priorstudies have shown that OT is stable in milk under high tem-perature, adverse pH and simulated gastric fluid condition [5]suggesting that absorption may occur eliciting undesired effects.Therefore, even if 0.5–1% OT is bioavailable through oral consump-tion of contaminated milk, neonates and children become the mostvulnerable groups. This issue is more important because the intakeof OT occurs during the neonatal and childhood period of life andmay affect central nervous system development, due to the leakynature of infant blood–brain barrier.

Although reports are available that OT regulates estrous cyclelength, follicle luteinization and ovarian steroidogenesis [10,11];continuous oral exposure of OT from early age has not been stud-ied. Therefore, the present study was carried out to investigate theanomalies in ovary and other organs following daily oral adminis-tration of OT to young immature rats. Initially, the serum proteinmarkers of ovulation were studied as this is an inflammatory pro-cess and the increase in inflammatory serum proteins are indicativeof ovulation [12]. This was supported by the enhanced levels ofPGE-2, an important mediator of ovulation process [13,14]. Fur-ther, the number of corpous leuteum (CLs) and the expression ofdifferent proteins related to intermediate steps of ovulation viz.granulosa cells survival and folliculogenesis (p-EGFR, ERK1/2 and

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M. Mishra et al. / Reproductive Toxicology 40 (2013) 24– 34 25

GDF-9), matrix deposition (TSG-6, HAS-2), cumulus expansion andfollicle rupture (COX-2) were also studied [15–19].

2. Materials and methods

2.1. Chemicals

Standard OT was purchased from Sigma Chemicals Co. (St. Louis, MO). [3H]OT (oxytocin, tyrosyl-2,6-3H) (30–60 Ci/mmol) was a product of PerkinElmer(Waltham, MA). Primary antibodies [phosphorylated epidermal growth factorreceptor (pEGFR), epidermal growth factor receptor (EGFR), cyclooxygenase-2(COX-2), phosphorylated extracellular signal regulated kinase 1/2 (pERK1/2), extra-cellular signal regulated kinase 1/2 (ERK1/2), growth differentiation factor-9(GDF-9), hyaluranic acid synthase-2 (HAS-2), TNF stimulating gene-6 (TSG-6), B-cell lymphoma 2 (Bcl2), poly ADP ribose polymerase (PARP) and �-actin] andHRP-conjugated secondary antibodies (anti rabbit, anti mouse and anti goat)were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). [3H] thymi-dine (6.7 Ci/mmol) was purchased from PerkinElmer (Waltham, MA). PGE-2, pERKinhibitor (PD98059) and PGE-2 inhibitor (Arachidonyl trifluro methyl ketone)were procured from Cayman Chemicals (Ann Arbor, MI). OT antagonist [d(CH)2

51Tyr(Me)2Arg8 vasopressin] (OTA) was purchased from Tocoris Bioscience (Bristol,UK). All other chemicals and reagents used were of the highest purity commerciallyavailable.

2.2. In vitro evaluation of OT internalization in intestinal epithelial cell line(IEC-6) cells

2.2.1. Cell cultureIEC-6 cells (NCCS, Pune, India) were routinely cultured in 25-cm2 flasks at

37 ◦C under the influence of 5% CO2 and 95% humidified atmosphere in Dulbecco’sModified Eagles Medium (DMEM) having 100 IU/ml penicillin and 100 �g/ml strep-tomycin, supplemented with 10% fetal bovine serum (FBS) and 0.25% insulin. Cellswere detached from the flasks with trypsin/EDTA after reaching the sub confluentstate and cell viability was checked. The cell viability was performed by 1:1(V/V)dilution of the cell suspension with 0.4% trypan blue solution. This mixture wasloaded onto the chambers of a haemocytometer and the number of stained cellsand total number of cells were counted. The numbers of unstained cells representviable cells and were calculated as a percentage of total cells. The cells were platedin 12 well plates for each experiment.

2.2.2. Internalization assayFor studying the internalization of OT, the IEC-6 cells were seeded in 12 well

plates at a density of 1 × 105 cells/2 ml. After 24 h of plating the cells were treatedwith 2 �Ci of [3H] OT for 15 min and 24 h, respectively. After completion of incuba-tion, the cells were gently scraped and harvested in glass fiber filters. These filterswere transferred to scintillation cocktail W (Sisco Research Laboratories Pvt. Ltd.,Mumbai, India) and counts were recorded on Hewlett–Packard � counter (Palo Alto,CA). For studying the localization of internalized OT, the cells were homogenizedand sub-cellular fractionation was carried out by differential centrifugation [20].The radioactivity was counted in sub-cellular fractions in � counter as describedabove.

2.3. Animals and treatment protocol for oral administration

Healthy female Wistar rat pups (7-day-old) along with their mother wereobtained from the animal breeding colony of the Indian Institute of ToxicologyResearch (IITR) Lucknow, India. The animals were acclimated under standard lab-oratory conditions for 3 days prior to the experiment. Animals were housed inair-conditioned room in plastic cages and maintained at 22 ± 2 ◦C under standardlaboratory conditions of light/dark cycle (12–12 h) and had free access to food andwater ad libitum. Pups (12 ± 3 g) were randomly divided into three groups of 15 each.Oral intubation of freshly prepared OT (1 and 10 ng) was given daily for 25 days (fromd10 to d35). Pups were separated from dams at 22 days of age. Doses of OT were giventhrough soft plastic tube faced cannula to avoid damage in buccal cavity of pups.The control group received daily oral intubation of vehicle. During the treatmentschedule, the animals were weighed twice weekly. After 25 days of treatment, ratswere monitored daily for estrus stage through vaginal smear and sacrificed in themorning of estrus by cervical dislocation according to the guidelines for the care anduse of laboratory animals of IITR. All the major organs including liver, lungs, kidney,heart, brain and ovaries from each animal were dissected out and weighed. Threeanimals from each group were sacrificed before estrus and ovaries were dissectedout to check the effects of oral OT on immature ovaries (before ovulation).

2.4. Serum protein levels and profile

Total protein and albumin (A) contents in serum was assayed by commercial kit(Accurex Biomedical Pvt. Ltd., Mumbai, India). Globulins (G) and A/G ratio werecalculated from these values and expressed as g/dl. Profiling of serum proteinswas carried out on electrophoresis using a pre-formed SDS-PAGE gel from Sebia

(Gwinnett, GA) and densitometric scanning was performed on Phoresis softwarefrom Sebia (Gwinnett, GA).

2.5. Quantification of ovarian follicles

Quantification of ovarian follicle in ovaries of animals was performed accord-ing to the procedure described earlier [21]. Briefly, ovaries were fixed in 4%paraformaldehyde, dehydrated, and embedded in paraffin. To count the numbersof follicles, paraffin-embedded ovaries were serially sectioned at 5-�m thicknessand stained with hematoxylin for morphological observation. Ovarian follicles atdifferent developmental stages were counted in all sections of an ovary, based onthe well-accepted protocol established by Pedersen and Peters [22].

2.6. Histopathological processing

Liver, brain, kidney, lung, heart, uterus and ovaries were washed in cold salinesoak dried on filter paper and weighed. A portion of organ was fixed in 10% bufferedformalin and embedded in paraffin. Sections of 5 �m thickness were cut and stainedwith hematoxylin and eosin for microscopic examination [23].

2.7. Prostaglandin E2 (PGE2) assay

The level of PGE2 in ovaries was determined by enzyme-linked immunoassayaccording to the manufacturer’s manual (Cayman Chemical Co, Ann Arbor, MI). Totalprotein concentration in the ovary was determined with the bicinchoninic acid(BCA) protein assay kit (Thermo Fisher Scientific, Rockford, IL). PGE2 values wereexpressed as picogram PGE2 per milligram tissue protein.

2.8. Western blot analysis

For western blot analysis, whole cell lysates were prepared from 6 pooledovaries collected from control and 10 ng OT treated groups using lysis buffer[10 mM Tris–HCl (pH 7.4), 100 mM NaCl, 1 mM EGTA, 1 mM EDTA, 50 mM NaF,1 mM DTT, 20 mM sodium pyrophosphate (Na4P2O7), 2 mM sodium orthovanadate(Na3VO4), 1% Triton X-100, 10% glycerol, 0.1% SDS, 0.5% sodium deoxycholate andprotease inhibitor cocktail tablets]. Ovarian cell lysates were electrophoresed on 6%SDS-polyacrylamide gel for PARP, pEGFR and EGFR protein, while rest of the pro-teins like COX-2, pERK1/2, ERK1/2, GDF-9, HAS-2, TSG-6, Bcl-2, and �-actin wereelectrophoresed on 10% SDS-polyacrylamide gel. Separated proteins were elec-trophoretically transferred onto polyvinylidene difluoride (PVDF) membranes. ThePVDF membranes were blocked with 1% BSA in TBS-T [20 mmol/L Tris, 150 mmol/LNaCl (pH 7.5), 0.02% Tween 20] for 1 h at room temperature and further incu-bated with different antibodies of EGFR (1:1000), pEGFR (1:500), pERK 1/2 (1:1000),ERK1/2 (1:1000), COX-2 (1:1000), GDF-9 (1:1000), TSG-6 (1:1000), HAS-2 (1:1000),and �-actin. The detection of these proteins was carried out by incubating withcorresponding secondary antibodies. Signals were detected by the enhanced chemi-luminescence substrates supplied in Electro chemiluminescence kit (Thermo FisherScientific, Rockford, IL).

2.9. Granulosa cell culture

Granulosa cells were harvested from estradiol primed intact immature (day25) rats as described by Carlone and Richards [24]. Briefly, cells were harvestedby aspiration and cultured at a density of 5 × 104 cells per 0.5 ml of medium (1% FBScontaining DMEM F-12 medium with penicillin and streptomycin) in each well of 48well plates and kept overnight. After 24 h the medium was removed, the attachedcells were washed with serum free medium and kept overnight followed by additionof OT (10 nM), OTA (19 �M), pERK1/2 inhibitor (10 �M), PGE-2 (1 �M) and PGE-2inhibitor (25 �M) in 0.5 ml of serum free DMEM F-12 medium. The concentrationsof these inhibitors were similar to those used by other investigators [25–27]. After24 h of treatment, cells were analyzed for proliferation and pERK1/2 expression bythymidine incorporation and western blot analysis, respectively.

2.10. Measurement of [3H] thymidine incorporation in granulosa cells

To measure cell proliferation rate, 5 × 104 granulosa cells were seeded in eachwell of 48 well plates in 0.5 ml of complete medium in triplicate with OT, OTA,pERK1/2 inhibitor, PGE-2 and PGE-2 inhibitor as mentioned above in Section 2.9.[3H] thymidine (2 �Ci) was added to each well, 18 h prior to the completion of 24 hof incubation time [28,29]. The cells were collected with the help of harvester andincorporated radioactivity was measured in a liquid scintillation counter (PackardBioscience Company, Meriden, CT).

2.11. Statistical analysis

All results were expressed as the mean ± standard error (SE), as indicated inthe tables. Statistical analysis of variance was carried out using one way ANOVAfollowed by Dunnet post hoc test [30]. A value of P < 0.05 was used as the level ofsignificance.


26 M. Mishra et al. / Reproductive Toxicology 40 (2013) 24– 34

3. Results

3.1. OT internalization in IEC-6 cells

The [3H] OT in IEC-6 cells was found to increase about 26-and 30-fold following 15 min and 24 h of incubation, respectively(Fig. 1A). The internalization of [3H] OT was monitored in differentfraction of IEC-6 cells incubated with OT for 15 min and 24 h. Theresults showed that maximum radioactivity was retained in plasmamembrane (PM) fraction at 15 min while after 24 h the maximumradioactivity was found in mitochondrial and microsomal fractionwhen the data was expressed as counts/mg protein (Fig. 1B). Whenthe data was expressed as percent of total radioactive counts, 0.07%and 0.03% of radioactive OT uptake was observed in respectivemitochondrial and microsomal fraction after 24 h, while 0.002% ofradioactivity was noticed in plasma membrane (Fig. 1C).

3.2. Effect of oral intubation of OT to rat pups on body weight andovarian weight

The trend in body weight of rats in control and OT exposure for25 days is given in Fig. 2A. Significant reduction (25% and 31%) inbody weight was observed in rats treated with 1 ng and 10 ng OTfor 25 days. Weight of ovaries was significantly increased (10–18%)in 1 ng and 10 ng OT treated groups (Fig. 2B).

3.3. Changes in serum protein levels and profile following OTintubation

Total protein content in serum was significantly increased (28%)following 1 ng and 10 ng OT treatments to animals (Fig. 3A). OT (1and 10 ng) treatment to pups for 25 days resulted in significantincrease (27–28%) in globulin content while albumin content wasnot altered in any of the OT treated groups (Fig. 3B and C). A/G ratiowas found to be decreased (25–30%) in OT (1 and 10 ng) treatedanimals (Fig. 3D). Further, there was no significant change in anyof the parameters in 0.1 ng OT treated rats (Fig. 3A–D).

Serum protein profiling through SDS-PAGE electrophoresisshowed no change in albumin content while increases wereobserved in all the forms of globulins (�1, �2 and � globulin) except� globulin in OT (1 and 10 ng) treated animals (Fig. 3E and F).

3.4. Histopathological observation of liver, brain, kidney, lung,heart, uterus and ovary of OT treated rats

Microscopic examination of liver, brain, heart, kidney, lung,spleen and uterus of OT (10 ng) treated animals for 25 days showednormal histological structure similar to that of control animals(Fig. 4A(i–vii)).

Histopathology of immature ovaries (before ovulation) (1 and10 ng) showed different stages of follicles and cystic dilation of

Fig. 1. Internalization of [3H] OT in IEC-6 cells. (A) [3H] OT in whole IEC-6 cells following incubation for 15 min and 24 h. (B) Internalization of [3H] OT (counts per mgprotein) in different fractions of IEC-6 cells. Each value represents mean ± SE (n = 3). *p < 0.05, significant when compared to respective control. (C) Internalization of percentradioactivity of [3H] OT in different fractions of IEC-6 cells. Percent radioactivity was derived from mean values of graph 1B.



Fig. 2. Effect of OT intubation to rat pups on body weight and ovarian weight. (A)Effect of OT on body weight. (B) Effect of OT on ovary weight. Each value representsmean ± SE (n = 15).*p < 0.05, significant when compared to control.

ovarian bursa and profuse interstitial edema leading to enlarge-ment of ovaries (hypertrophied) along with greater number offollicles compared to control rats (Fig. 4B(i and ii)). Animals treatedwith OT (1 and 10 ng) for 25 days (after ovulation) showed greaternumber of CLs as compared to control animals (Fig. 4C(i and ii)).

3.5. Effect of OT incubation to rat pups on anti apoptotic proteinsBcl-2 and PARP expression

OT treatment to rat pups caused increased expression of Bcl-2 protein (2.3-fold) while PARP expression was substantiallydecreased (0.4-fold) [Fig. 4B(iii)].

3.6. Effect of oral OT intubation to rat pups on ovarian PGE-2 level

OT treatment (1 and 10 ng) to rat pups for 25 days resulted insignificant increase (41–74%) in ovarian PGE-2 levels (Fig. 5A).

3.7. Effect of OT on p-EGF receptor, pERK 1/2, COX-2, GDF-9,HAS-2, and TSG-6 levels in ovaries of rats

The levels of pEGFR in ovary was found to be enhanced (2.1-fold)following OT (10 ng) treatment to rats for 25 days when compared

to control (Fig. 5B). Furthermore, pERK1/2, which is involved inovulation process, was found to be enhanced (1.5-fold) followingOT intubation to rats. OT treatment also led to significant up reg-ulation of COX-2 (3.2-fold), GDF-9 (1.6-fold), HAS-2 (2.2-fold), andTSG-6 (2.4-fold) in the ovaries of rats (Fig. 5B).

3.8. Effect of OT, OTA, pERK1/2 inhibitor, PGE-2 and PGE-2inhibitor on OT induced granulosa cells proliferation

Morphological data on granulosa cells and thymidine uptakefollowing treatment with OT, OTA, pERK1/2 inhibitor, PGE-2 andPGE-2 inhibitor is shown in Fig. 6A and B. Treatment of gran-ulosa cells with OT caused increased proliferation along withattachment to the surface. However, treatment of OT inducedgranulosa cells with OTA or pERK1/2 inhibitor or PGE-2 inhibitorshowed decreased proliferation activity (Fig. 6A). These morpho-logical results were substantiated with [3H] thymidine uptakeexperiments wherein OT treatment caused significant enhance-ment of thymidine as compared to control, while OTA treatment toOT induced cells resulted in significant reduction in radioactivityuptake when compared to cells treated with OT. Similarly, treat-ment of OT induced cells to pERK1/2 inhibitor or PGE-2 inhibitorresulted in significant decrease in thymidine uptake compared toOT treated cells (Fig. 6B). Further, OT induced cells treated withpERK1/2 inhibitor along with PGE-2 resulted in significant increasein thymidine uptake compared to OT + pERK1/2 inhibitor treatedcells (Fig. 6C). Western blot analysis of pERK1/2 in granulosa cellsin the presence of inhibitors is shown in Fig. 6C. Treatment of cellswith OT resulted in enhanced (1.6-fold) expression of pERK1/2.However, OTA or pERK1/2 inhibitor treatment to OT induced cellscaused decrease in pERK1/2 expression when compared to OTtreated cells. Treatment of OT induced cells with pERK1/2 inhibitorin presence of PGE-2 resulted in decrease expression of pERK1/2when compared to OT treated alone, while treatment with PGE-2inhibitor to OT induced cells showed no change in pERK1/2 whencompared to OT exposure alone (Fig. 6C).

4. Discussion

A 25-day (d10–d35) oral exposure of OT was investigated inyoung female rats to understand ovarian anomalies. The centralfinding of this article demonstrates that early age oral exposure toOT has a greater effect on the ovary and leads to increased ovulationin female rats. The animals treated with OT showed increased ovar-ian weight. Corbin and Schottelius [31] also found increased ovarianweight following OT injection to postnatal rats. Histopathologicalanalysis of ovaries before estrus (ovulation) showed a higher num-ber of follicles, which clarifies the reason for higher ovarian weightfollowing OT exposure. The increased numbers of follicles were fur-ther followed by an increased ovulation rate as obtained by highernumber of CLs in mature rats (on estrus). During follicular mat-uration only some of the matured follicles get ovulated while allothers led to atresis [32]. Therefore, this study suggest that earlyage oral OT exposure not only increases the follicle number butalso affects the mechanism through which selection of the oocyteswere taken for ovulation. Ovulation is an inflammation like processand the increase in inflammatory serum proteins are also indicativeof ovulation process [12]. Therefore, in the present study increasesin serum � globulin level obtained following OT exposure furthersupport our hypothesis of increased ovulation under the influenceof OT.

Ovulation is a directed and sequential process accompanied bybroad-spectrum proteolysis culminating in the follicular ruptureto release the matured oocyte. During the late stage of folliculo-genesis, a population of follicular granulosa cells form a cumulus



Fig. 3. Changes in serum protein levels and profile following OT intubation to rat pups. (A) Total protein, (B) globulin, (C) albumin, (D) A/G ratio, (E) densitometric scanning ofSDS-PAGE electrophoresis of serum from control and OT treated rats, (F) levels of albumin and various globulins in serum of control and OT treated rats. Each value representsmean ± SE (n = 3). *p < 0.05, significant when compared to control.



oophorus structure in the periphery of the ovum which is heldtogether by a gap junction network and subsequently by an exten-sive matrix by ovulatory signal. The preovulatory deposition ofcumulus oophorus matrix causes volumetric enlargement of cumu-lus oocyte complex called as cumulus expansion and dissociationof cumulus oocyte complex from the follicle wall, accompaniedby resumption of meiosis that is characterized by germinal vesi-cle breakdown. At the same time, ovulatory signal also stimulatesmatrix degrading protease secretion, which results in follicle wallrupture to extrude cumulus oocyte complex.

In the present study, we have observed the effect of early expo-sure of OT on different proteins related to intermediate processesof ovulation viz. granulosa cells survival, matrix deposition, cumu-lus expansion and follicle rupture. Further, we have also observedthat OT caused proliferation of granulosa cells under in vitro con-ditions, which involve enhanced pERK expression. We had takengranulosa cells because they are the structural basis of the ovaryand the cell population is mainly responsible for responses towardgonadotropins [33].

The mechanism of increased ovulation following oral adminis-tration of OT to rat pups may be due to higher expression of pEGFRand its downstream pERK1/2 protein indicating its facilitatory

effects toward ovulation. It has been reported that immaturetransgenic mice (Erk1/2gc−/−) treated with exogenous hormone(ovulatory signal, i.e. LH) show no ovulation, which suggests therole of pERK1/2 for ovulation [15,34]. However, the study of Rimoldiet al. [16] suggests that OT receptors can transactivate EGFR andthereby ERK1/2 sequentially using different signaling intermedi-ates. To understand the role of pERK1/2 in OT induced ovulationwestern blot analysis of pEGFR and pERK1/2 was performed. Theover expression of pEGFR and up regulation of pERK1/2 in ovaries ofOT treated female rats emphasizes the possible role of pERK1/2 inOT induced ovulation. pERK1/2 is a key protein which can regulatedifferent intermediate proteins of ovulation [34].

The increased level of ovarian PGE-2 following oral OT expo-sure further supports our data of increased ovulation as studies inrats have indicated that PGE2 has a stimulatory effect on ovula-tion [13]. Other functions of the ovary related to ovulation maybe affected by oral OT exposure as shown by increased expres-sion of HAS-2 and TSG-6, which plays a crucial role in matrixdeposition (mucification) [17,35]. Increased expression of proteinlike COX-2 by OT, which is related to cumulus oocyte complexexpansion process and proteolysis of follicular wall during ovu-lation support the next step of ovulation [14,18]. Increased GDF-9

Fig. 4. Histopathological observation of different organs of OT treated rats. (A) Histopathological observation of liver (i), kidney (ii), heart (iiii), lung (iv), brain (v, vi) anduterus (vii) of OT treated young rats. (B) Quantification of total follicles in H&E staining of ovarian sections (before ovulation) of OT treated rats (i and ii), western blot ofantiapoptotic protein Bcl-2 and PARP in ovarian lysates (before ovulation) (iii). *p < 0.05, significant when compared to control. (C) Quantification of Corpus leuteum in H&Estaining of ovarian sections (after ovulation) of OT treated rats (i and ii).



Fig. 4. (continued)

protein (oocyte secreted factor) in the ovaries of OT treated ratssuggests that follicular maturation and ovulation may be enhancedas GDF-9 has been shown to be an important factor for follicu-logenesis and ovulation efficiency [19]. These proteins in ovarieswere found to be substantially over expressed following OT expo-sure to rat pups suggesting enhanced matrix deposition in theovaries during cumulus expansion followed by rapid proteoly-sis of follicular wall which ultimately led to increased ovulationprocess.

To further validate the involvement of pERK1/2 protein in theprocess of OT induced enhanced ovulation, we performed exper-iments in primary granulosa cells and investigated the effect ofPGE-2 and inhibitors of OT, pERK1/2 and PGE-2 on proliferation andkey protein pERK1/2 expression. The results indicate that the effectof OT on granulosa cells is mediated through pERK1/2, as pERK1/2inhibitor in presence of OT caused decreased proliferation of gran-ulosa cells. PGE-2, the main effecter molecule, works downstreamto pERK1/2, as PGE-2 in the presence of OT and pERK1/2 inhibitorwas capable of cell proliferation. These results confirmed that OTthrough induction of pERK1/2 caused the secretion of PGE-2, whichultimately led to granulosa cell proliferation and thereby increas-ing cell survival. The increased level of ovarian PGE-2 following OTtreatment to rat pups further supports the in vitro data.

The putative mechanism of increased ovulation following OTexposure to female rat pups may be through activation of an OTreceptor which transactivates the pEGFR which thereby activatesthe ERK1/2, the key protein for ovulation which further regulatesthe secretion of PGE-2 and expression of COX-2, GDF-9, HAS-2 andTSG-6 proteins and thereby granulosa cell survival, differentiation,matrix deposition, and cumulus expansion processes as shown inFig. 7.

Increased internalization of radioactive OT in IEC-6 cells furthersupports the efficacy of orally administered OT, as intestinal cellsare equipped with OT receptors which may facilitate the absorp-tion of OT in intestine [36]. In spite of the fact that during earlylife (till 21 postnatal days in rat) intestinal permeability is higheras compared to adult [37], our in vitro data provided evidence forinternalization of [3H] OT in 15 min which is further enhanced withincubation time (24 h). Conti et al. [38] showed that stimulationof G protein-coupled OT receptors (OTRs) leads to internalizationof ligand–receptor inside intracellular compartments by endocyto-sis which further supports our present results. Cell fractionizationstudies on IEC-6 cells following incubation with [3H] OT for 15 minand 24 h indicated that internalization is receptor mediated asradioactivity was found to be higher in the plasma membrane frac-tion after 15 min of [3H] OT incubation and remained constant evenafter 24 h incubation time. Further, with an increase in incubationtime (24 h) concentrations of OT in mitochondrial and microsomalfractions were enhanced by 24- to 30-fold suggesting that internal-ization of cell-surface bound radioactive OT begins immediatelyfollowing the receptor ligand binding. After internalization, OTreaches to its target organs and can cause their altered manifes-tations, i.e. induced ovulation. Our surveillance study showed thatthe extent of OT adulteration in milk is up to 100%, while basedon milk consumption in children per day, the doses of OT usedin the present study assume a great significance in exerting theiranomalous response in ovary.

The overall results suggest that oral OT exposure (through OTcontaminated milk) since early childhood may not show alterationin classical toxicological parameters but its target organ, the ovary,is a matter of concern as it enhances ovulation which may haveunwanted consequences that needs to be explored further.



Fig. 5. Effect of oral intubation of OT to rat pups on different ovulation markers. (A) PGE-2 level. (B) Western blots of pEGFR, pERK1/2, COX-2, GDF-9, HAS-2, and TSG-6proteins. Densitometric values were shown above the bands in terms of folds considering control as 1.



Fig. 6. Effect of OT, OTA, pERK1/2 inhibitor, PGE-2 and PGE-2 inhibitor on granulosa cell proliferation and pERK1/2 expression. (A) Morphological observation of granulosacells following OTA, pERK1/2 inhibitor, pERK1/2 inhibitor + PGE-2 and PGE-2 inhibitor treatment for 24 h on OT induced proliferation. *p < 0.05, significant when comparedto control group. **p < 0.05, significant when compared to OT alone treated group. ***p < 0.05, significant when compared to OT + pERK1/2 inhibitor treated group. (B) [3H]Thymidine uptake of granulosa cells following OTA, pERK1/2 inhibitor, pERK1/2 inhibitor + PGE-2 and PGE-2 inhibitor for 24 h on OT induced proliferation. (C) Western blotof pERK1/2 in OT, OT + OTA, OT + pERK1/2 inhibitor, OT + pERK1/2 inhibitor + PGE-2 and OT + PGE-2 inhibitor treated (24 h) granulosa cells.



Oxytoc in

OTR

pEGFR

pERK1/2

COX-2 Matrix deposi tion prot ein

TSG-6 & HAS -2 Oocyt e fa cto r

(GDF -9)

Ovulat ion

PGE-2

Fig. 7. Mechanism of OT induced enhanced ovulation in the ovaries of rats.

Conflict of interest statement

The authors declare that there are no conflict of interest.

Acknowledgements

We are grateful to the Director of our institute for his keeninterest in the study. One of us (M. M.) is thankful to Council ofScientific and Industrial Research (CSIR)/University Grant Commis-sion (UGC), New Delhi for the award of Senior Research Fellowship.Thanks are due to Mr Ashish Yadav for checking the manuscript.Financial assistance of CSIR-INDEPTH Project-BSC 0111 is gratefullyacknowledged. The manuscript is IITR communication # 3063.

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Anomalies in ovary following oral exposure to oxytocin: Mechanistic studies

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