Elsevier

Reproductive Toxicology

Volume 40, September 2013, Pages 24-34
Reproductive Toxicology

Anomalies in ovary following oral exposure to oxytocin: Mechanistic studies

https://doi.org/10.1016/j.reprotox.2013.05.004Get rights and content

Highlights

  • 25 days oral OT exposure to 10 days old pups resulted in increased ovulation.

  • OT showed increase in ovarian weight and total number of follicles and CLs.

  • OT induced ovulation may involved up regulation of pEGFR and pERK1/2.

  • OT also up regulate the HAS-2 & TSG-6, COX-2 and GDF-9 proteins.

Abstract

Ovarian anomalies following oral oxytocin (OT) (1 and 10 ng/100 μl) 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 cattle thereby causing oral exposure to human population from early age. OT exposure resulted in increased ovarian weight, γ globulin, total number of follicles, and number of corpus luteum (CLs); indicating higher ovulation. The mechanism may involve over-expression of pEGFR followed by downstream pERK1/2 and subsequently increased ovarian PGE-2 along with enhanced COX-2, HAS-2 & TSG-6 (matrix deposition proteins) and GDF-9 (oocyte factor) proteins, suggesting that oral exposure of OT may affect the physiology and function of the ovary. Further, in vitro studies showed increased internalization of OT in IEC-6 cells which further supports that orally administered OT may cause altered manifestations as shown above following internalization in mucosal membrane.

Introduction

Oxytocin (OT), a biological hormone, has a physiological role of performing specific functions at different stages of life. For example OT stimulates labor by uterine contraction and has been implicated as a drug during delivery cases [1]. Under normal physiological conditions in lactating mothers, OT causes contraction of the myoepithelial cells which surround the milk alveoli in the mammary gland for milk let-down [2].

In the Indian subcontinent, OT ampules, commercially known as pitocin or syntocinon, are indiscriminately used to enhance milk let-down following intramuscular injections to cattle. The exogenous supplementation of OT is absorbed in blood thereby increasing the concentration (4- to 5-fold) and is retained for at least 2 h following injection to animals [3]. It has been presumed that due to small size of OT (1 kDa) there is a possibility that it crosses the blood–milk barrier reaching into the milk and thereby causing changes in the permeability of tight junctions of mammary gland [4]. Our prior study has shown the presence of OT in milk samples [5] which is substantiated by the work of Takeda et al. [6] wherein intraperitoneal injection of radiolabelled OT given to rat dams transfer to the plasma of neonates through milk.

A majority of peptides are known to be absorbed with less bioavailability (0.5–1%) [7]. This may increase according to the conditions found in the gastrointestinal (GI) tract [7], [8], [9]. Our prior studies have shown that OT is stable in milk under high temperature, 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 consumption of contaminated milk, neonates and children become the most vulnerable groups. This issue is more important because the intake of OT occurs during the neonatal and childhood period of life and may affect central nervous system development, due to the leaky nature of infant blood–brain barrier.

Although reports are available that OT regulates estrous cycle length, follicle luteinization and ovarian steroidogenesis [10], [11]; continuous oral exposure of OT from early age has not been studied. Therefore, the present study was carried out to investigate the anomalies in ovary and other organs following daily oral administration of OT to young immature rats. Initially, the serum protein markers of ovulation were studied as this is an inflammatory process and the increase in inflammatory serum proteins are indicative of ovulation [12]. This was supported by the enhanced levels of PGE-2, an important mediator of ovulation process [13], [14]. Further, the number of corpous leuteum (CLs) and the expression of different proteins related to intermediate steps of ovulation viz. granulosa cells survival and folliculogenesis (p-EGFR, ERK1/2 and GDF-9), matrix deposition (TSG-6, HAS-2), cumulus expansion and follicle rupture (COX-2) were also studied [15], [16], [17], [18], [19].

Section snippets

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 factor receptor (pEGFR), epidermal growth factor receptor (EGFR), cyclooxygenase-2 (COX-2), phosphorylated extracellular signal regulated kinase 1/2 (pERK1/2), extracellular signal regulated kinase 1/2 (ERK1/2), growth differentiation factor-9 (GDF-9), hyaluranic acid synthase-2

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 different fraction of IEC-6 cells incubated with OT for 15 min and 24 h. The results showed that maximum radioactivity was retained in plasma membrane (PM) fraction at 15 min while after 24 h the maximum radioactivity was found in mitochondrial and microsomal fraction when the data was expressed as counts/mg protein (

Discussion

A 25-day (d10–d35) oral exposure of OT was investigated in young female rats to understand ovarian anomalies. The central finding of this article demonstrates that early age oral exposure to OT has a greater effect on the ovary and leads to increased ovulation in female rats. The animals treated with OT showed increased ovarian weight. Corbin and Schottelius [31] also found increased ovarian weight following OT injection to postnatal rats. Histopathological analysis of ovaries before estrus

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 keen interest in the study. One of us (M. M.) is thankful to Council of Scientific and Industrial Research (CSIR)/University Grant Commission (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 gratefully acknowledged. The manuscript is IITR communication # 3063.

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