Contrast-Enhanced Ultrasonography (CEUS) in Imaging of the Reproductive System in Dogs: A Literature Review.

The use of contrast-enhanced ultrasound (CEUS) has been widely reported for reproductive imaging in humans and animals. This review aims to analyze the utility of CEUS in characterizing canine reproductive physiology and pathologies. In September 2022, a search for articles about CEUS in canine testicles, prostate, uterus, placenta, and mammary glands was conducted on PubMed and Scopus from 1990 to 2022, showing 36 total results. CEUS differentiated testicular abnormalities and neoplastic lesions, but it could not characterize tumors. In prostatic diseases, CEUS in dogs was widely studied in animal models for prostatic cancer treatment. In veterinary medicine, this diagnostic tool could distinguish prostatic adenocarcinomas. In ovaries, CEUS differentiated the follicular phases. In CEH-pyometra syndrome, it showed a different enhancement between endometrium and cysts, and highlighted angiogenesis. CEUS was shown to be safe in pregnant dogs and was able to assess normal and abnormal fetal-maternal blood flow and placental dysfunction. In normal mammary glands, CEUS showed vascularization only in diestrus, with differences between mammary glands. CEUS was not specific for neoplastic versus non-neoplastic masses and for benign tumors, except for complex carcinomas and neoplastic vascularization. Works on CEUS showed its usefulness in a wide spectrum of pathologies of this non-invasive, reliable diagnostic procedure.

Characterization of Testicular Tumor Lesions in Dogs by Different Ultrasound Techniques.

In this retrospective study, we assessed the accuracy of different blood flow imaging in diagnosing testicular tumor types in dogs. We recruited 27 dogs with leydigomas (14), seminomas (eight), sertoliomas (six), and mixed cells (five) confirmed histopathologically. In intact dogs, Pampiniform plexus and marginal arteries were scanned through pulsed Doppler. Blood flow and presence of intralesional/perilesional arteries were assessed by color and power Doppler, B-flow, and contrast-enhanced ultrasound. Tumor types did not differ by B-Mode ultrasonography characters. Pampiniform and testicular arteries of sertoliomas had higher (p < 0.05) pulsatility and resistive indexes. The proportion of leydigomas with a perilesional and/or perilesional/intralesional blood flow pattern detected by color and pulsed Doppler and B-flow was higher (p < 0.05) than that of the other tumors counted together. This resulted in a sensitivity of 81.8%, 83.3%, and 85.7%, a specificity of 76.5%, 56.3%, and 73.7%, and a correct classification rate of 78.6%, 67.9%, and 78.8%, respectively. While contrast enhanced ultrasound was highly effective in detecting all tumors, qualitative and quantitative parameters did not contribute to their differential diagnosis. In conclusion, results indicate that different testicular tumor types of dogs have subtly different vascular patterns, a condition that could help in identifying leydigomas.

Nerve Growth Factor (NGF) and Animal Reproduction.

Stimuli that lead to the release of gonadotropin-releasing hormone (GnRH) and pituitary gonadotropins and, consequently, ovulation in mammals fall into two broad categories. In the first, high plasma oestrogen concentrations induce the events that trigger ovulation, a characteristic of spontaneous ovulators. In the second, nerve stimuli occurring during mating reach the hypothalamus and trigger the release of GnRH and ovulation with a neuroendocrine reflex that characterizes induced ovulators.In this review, we will give an overview of the distribution of NGF and its expression in the different tissues of the male accessory sex glands, the main sites of NGF production. Next, we will highlight the role of NGF in sperm function and its potential cryopreserving role in artificial insemination techniques. Finally, we will evaluate the functions of NGF in ovulation, particularly in induced ovulators. Overall, the information obtained so far indicates that NGF is widely distributed in organs that regulate the reproductive activity, in both males and females. In spontaneous ovulators, NGF exerts mainly a luteotrophic action, while, in induced ovulators it is the main ovulation-inducing factor. A better understanding of the role of NGF in reproduction would be of great interest, since it could help finding innovative therapeutic aids to improve mammalian fertility.

Current Knowledge on the Multifactorial Regulation of Corpora Lutea Lifespan: The Rabbit Model.

Our research group studied the biological regulatory mechanisms of the corpora lutea (CL), paying particular attention to the pseudopregnant rabbit model, which has the advantage that the relative luteal age following ovulation is induced by the gonadotrophin-releasing hormone (GnRH). CL are temporary endocrine structures that secrete progesterone, which is essential for maintaining a healthy pregnancy. It is now clear that, besides the classical regulatory mechanism exerted by prostaglandin E2 (luteotropic) and prostaglandin F2α (luteolytic), a considerable number of other effectors assist in the regulation of CL. The aim of this paper is to summarize our current knowledge of the multifactorial mechanisms regulating CL lifespan in rabbits. Given the essential role of CL in reproductive success, a deeper understanding of the regulatory mechanisms will provide us with valuable insights on various reproductive issues that hinder fertility in this and other mammalian species, allowing to overcome the challenges for new and more efficient breeding strategies.

The Role of Resveratrol in Mammalian Reproduction.

Resveratrol is one of the most investigated natural polyphenolic compounds and is contained in more than 70 types of plants and in red wine. The widespread interest in this polyphenol derives from its antioxidant, anti-inflammatory and anti-aging properties. Several studies have established that resveratrol regulates animal reproduction. However, the mechanisms of action and the potential therapeutic effects are still unclear. This review aims to clarify the role of resveratrol in male and female reproductive functions, with a focus on animals of veterinary interest. In females, resveratrol has been considered as a phytoestrogen due to its capacity to modulate ovarian function and steroidogenesis via sirtuins, SIRT1 in particular. Resveratrol has also been used to enhance aged oocyte quality and as a gametes cryo-protectant with mainly antioxidant and anti-apoptotic effects. In males, resveratrol enhances testes function and spermatogenesis through activation of the AMPK pathway. Furthermore, resveratrol has been supplemented to semen extenders, improving the preservation of sperm quality. In conclusion, resveratrol has potentially beneficial effects for ameliorating ovarian and testes function.

Reproductive Traits of an Invasive Alien Population of Grey Squirrel (Sciurus carolinensis) in Central Italy.

The reproductive cycle of an invasive alien Italian grey squirrel population was studied to understand its adaptation and limit its spread, in order to conserve the autochthonous red squirrel. Female and male genital traits were evaluated throughout the reproductive cycle, including the ovary, uterus, testicle, epididymis, seminiferous tubule morphometry, and germinative epithelium histology. Moreover, individual female fecundity was determined by counting uterine scars. Ovary width and uterus weight, length, and width reached their highest values in the luteal and pregnancy phases. On conducting a histological evaluation of the testicular germinal epithelium, four morphotypes related to the different reproductive phases of the male squirrels were identified: immature, pubertal, spermatogenesis, and regressive. Testicle and epididymis weights and seminiferous tubule diameters reached their largest values during spermatogenesis. Uterine scar analysis showed that 69% of the females had given birth to one or two litters, while 31% had no uterine scars. Litters were larger in the first breeding period than in the second; annual fecundity was 4.52 ± 1.88 uterine scars/female. Umbrian grey squirrels have adapted to their non-native range, showing two annual mating periods at times similar to those in their native range, and high reproductive success.

Kisspeptin/GnRH1 system in Leydig cells of horse (Equus caballus): Presence and function.

The objectives of this study were to evaluate in horse testes the expression of kisspeptin (KiSS) and GnRH1 neuropeptides and their cognate receptors, KiSS1R and GnRH1R, as well as their action on testosterone, GnRH1, prostaglandin F2α (PGF2α), and PGE2 synthesis and cyclooxygenase 1 (COX1) and COX2 activity by Leydig cells in vitro. Testes were obtained from 9 sexually mature horses by surgical castration. Immunohistochemistry, evidenced the presence of KiSS, KiSS1R, GnRH, and GnRH1R in Leydig cells, whereas germinal and Sertoli cells were positive only for GnRH1. Transcripts for both neuropeptides and their cognate receptors were revealed in isolated Leydig cells by RT-PCR. Isolated and purified Leydig cells were in vitro cultured with agonists and antagonists of KiSS (KiSS-10 and KiSS-234, respectively) and GnRH1 (buserelin and antide, respectively). KiSS-10 and buserelin increased (P < 0.01) COX1 activity and testosterone and PGF2α basal secretion, while decreased (P < 0.01) that of PGE2. KiSS-10 and buserelin did not affect COX2 activity. GnRH1 basal production was increased (P < 0.01) by KiSS-10, but not by buserelin. Antide counteracted the KiSS and GnRH1 effects, whereas KiSS-234 influence only those of KiSS. Summarizing, the KiSS/GnRH1 system is present in horse Leydig cells and modulates their endocrine activity. In particular, the endocrine effects of KiSS are mediated by GnRH1, so suggesting that hypothalamic-like interaction between KiSS and GnRH1 occurs also in Leydig cells.

Effect of chelating and antioxidant agents on morphology and DNA methylation in freeze-drying rabbit (Oryctolagus cuniculus) spermatozoa.

Freeze-drying (FD) has been exhaustively tried in several mammalian species as an alternative technique to sperm cryopreservation, but few studies have been done in rabbits (Oryctolagus cuniculus). The main objective of this study was to compare the protective effect of various antioxidants added to EDTA medium on structural and functional components of FD rabbit spermatozoa and on their status of global DNA methylation. FD media used were composed of basic FD medium (10 mM Tris-HCl buffer and 50 mM NaCl) supplemented with either 50 mM EDTA alone (EDTA) or added with 105 µM of rosmarinic acid (RA, EDTA-RA) or 10 µM of melatonin (MLT, EDTA-MLT). The effect of each medium on the preservation of FD spermatozoon structure was evaluated with light and scanning electron microscopy (SEM). Global DNA methylation was quantified in all FD sperm samples as well as in fresh spermatozoa. Morphologically, fracture points were evidenced in the neck, mid and principal piece of the spermatozoon tail. No differences in spermatozoon fracture points were evidenced among FD treatments: intact spermatozoa were the largest (p < .01) category, whereas the most frequent (p < .01) injury was the neck fracture, resulting in tailless heads. At SEM, the head of spermatozoa showed a well-conserved shape and intact membrane in all treatments. DNA methylation status was the same in all FD treatments. In conclusion, supplementation of EDTA, EDTA-RA and EDTA-MLT during FD preserved rabbit sperm morphological integrity and methylation status as well. Therefore, the difficulty of getting viable offspring using FD semen is likely unrelated to the impact of the lyophilization process on DNA methylation and morphology of lyophilized spermatozoa.

Kisspeptin/kisspeptin receptor system in pseudopregnant rabbit corpora lutea: presence and function.

Kisspeptin (KiSS) and its related receptors (KiSS1R) have a critical role in the reproduction of mammals. The KiSS/KiSS1R system is expressed in numerous reproductive organs including the ovary. Here, we studied the expression of the KiSS/KiSS1R system and its functional role in rabbit corpora lutea (CL) at days 4 (early-), 9 (mid-), and 13 (late-stage) of pseudopregnancy. In vitro progesterone, prostaglandin (PG) F2α (PGF2α) and E2 (PGE2) productions and prostaglandin-endoperoxide synthase 1 (PTGS1) and 2 (PTGS2) activities were evaluated. Immune reactivity (IR) for KiSS and KiSS1R were detected in luteal cells at nuclear and cytoplasmic level at all luteal stage for KiSS and only at early- and mid-stage for KiSS1R; IR decreased from early- to later stages of pseudopregnancy. The KiSS-10 augmented progesterone and PGE2 and diminished PGF2α secretions by early- and mid-CL; KiSS-10 reduced PTGS2 activity at early- and mid-stages, but did not affect PTGS1 at any luteal stages. The antagonist KiSS-234 counteracted all KiSS-10 effects. This study shows that the KiSS/KiSS1R system is expressed in CL of pseudopregnant rabbits and exerts a luteotropic action by down-regulating PTGS2, which decreases PGF2α and increases PGE2 and progesterone.

Intra-ovarian dynamic blood flow in pseudopregnant rabbits during prostaglandin F2α-induced luteolysis.

In the present study, we evaluated the dynamic changes of intra-ovarian blood flow, by real-time colour-coded and pulsed Doppler ultrasonography, as well as the immunopresence of prostaglandin F2α (PGF2α) receptor (FP) and peripheral plasma progesterone concentrations in pseudopregnant rabbit after PGF2α treatments at either early- (4 days) and mid-luteal (9 days) stages. During the pre-treatment observation interval of one hour, the ovarian blood flows showed a fluctuating pattern. Independently of luteal stage, PGF2α administration caused a fourfold decline in the blood flow within 40 min that was followed 50 min later by a reactive hyperaemia that lasted several hours, while the resistive index showed an opposite trend. Twenty-four hour later, the blood flow was one half that measured before PGF2α injection. At day 4 of pseudopregnancy, PGF2α did not affect peripheral plasma progesterone concentrations, but at day 9, it caused functional luteolysis as progesterone levels declined 6 hr later to reach basal values after 24 hr. The changes in the ovarian blood flows of pseudopregnant rabbits receiving PGF2α were accompanied by simultaneous changes in the resistance index. This biphasic response in the blood flow and vascular resistances likely reflects reactive hyperaemia following vasoconstriction. By immunohistochemistry, strong positive immune reaction for FP was detected in the cytoplasm of endothelial cells of ovarian arteries, veins and capillaries. In conclusion, these results suggest that PGF2α could acutely regulate the ovarian blood flow of pseudopregnant rabbits, even if there is no evidence of a blood flow reduction anticipating luteolysis.

Contrast-enhanced ultrasonography of maternal and fetal blood flows in pregnant bitches.

We evaluated the potential usefulness of CEUS to assess fetal-maternal circulation during pregnancy in dogs. Nine bitches were examined at 23, 30, and 45 days of gestation using an ultrasound machine (LOGIQ E9) and SonoVue® contrast media as echo-signal enhancer. Qualitative and quantitative evaluation of contrast enhancement patterns of uterine artery and utero/placental vessels were performed on recorded images. Independently of the gestational periods, the qualitative evaluation showed the initial wash-in phase from the first appearance of the uterine artery to the rapid distribution in embryonic vesicles or placenta to the progressive washout, whilst there was no enhancement of either embryos or fetuses in any bitch. Independent of gestational age, parameters derived from quantitative analysis of time intensity-curves of contrast enhancement (peak intensity, time to peak, rise time, washout) did not vary between proximal placenta, distal placenta, and uterine artery. With the progression of gestation, AUC values did not change in both proximal and distal placenta, but in the uterine artery it was lower (P ≤ 0.05) at day 30 than at day 23 (464.8 ±â€¯16.1 vs.596.4 ±â€¯28.1, respectively). In conclusion, CEUS appears to safely permit evaluation of the maternal and fetal vessels in the first two third of gestation, without any clinically relevant adverse effects. Further studies in a larger number of bitches in different stages of pregnancy are needed to establish standard parameters for normal pregnancies that can be used to detect abnormalities of pregnancy.

New insights on a NGF-mediated pathway to induce ovulation in rabbits (Oryctolagus cuniculus).

To investigate the ovulatory mechanisms triggered by raw semen (RS) in rabbits, we examined the expression of nerve growth factor (NGF)-a supposed ovulation-inducing factor (OIF)-and cognate receptors in anterior pituitary, ovary, and cervix as well as plasma NGF and luteinizing hormone (LH) concentrations. Six does/group were sham-inseminated with sterile saline (PBS), naturally mated (NM), inseminated with RS alone or after lumbar anesthesia (ARS), or treatment with COX inhibitors (CIRS). Immunohistochemistry revealed positive signals for NGF and receptors in all tissues. RT-PCR confirmed the presence of the target transcripts in the same tissues, except NTRK1 in the cervix. Circulating NGF concentrations rose 3- to 6-fold (P < 0.01) 15 min after semen deposition into the genital tract of NM, RS, and ARS rabbits and remained sustained thereafter. Circulating NGF was 4-fold lower (P < 0.01) in CIRS than in RS does indicating that NGF is mainly synthesized by the uterus. A concomitant rise of LH and NGF concentrations was found in 83.3%, 50.0%, and 16.7% of NM, RS, and CIRS does, respectively, but not in ARS (despite high NGF circulating levels). Seminal plasma NGF concentration was 151.9 ± 9.25 µg/mL. The ovulatory responses were 0%, 83.3%, 66.7%, 16.7%, and 0% in PBS, NM, RS, ARS, and CIRS groups, respectively. Present data confirm that, although RS may induce ovulation via endocrine mechanisms through binding to NGF receptors in the ovary, a novel OIF-mediated neural mechanism facilitates ovulation in rabbits.

Contrast-enhanced ultrasound complements two-dimensional ultrasonography in diagnosing gallbladder diseases in dogs.

Gall-bladder diseases are common in dogs and two-dimensional ultrasonography is a current standard method for diagnosis and treatment planning. However, findings from this modality can be nonspecific. The aim of this retrospective, case series study was to describe conventional and contrast-enhanced ultrasound (using SonoVue® ) findings in a group of dogs with histologically confirmed gall bladder disease. A total of 65 dogs were included. Branchlike, heterogeneous, and homogeneous contrast enhancement of echogenic intraluminal mass-forming lesions was a contrast-enhanced ultrasound characteristic of polypoid lesions due to cystic mucosal hyperplasia of the gallbladder and/or tumor, which had different wash-in and washout characteristics. In dogs with mobile or immobile biliary sludge or mucocele, the echogenic intraluminal masses remained unenhanced. A double rim mark or enhancement defect in the gallbladder wall was a characteristic of edema or necrosis/rupture of the wall, respectively. Conventional ultrasonography correctly identified biliary sludge or mucocele in 36/37 dogs, cholecystitis/edema in 44/47 dogs, necrosis/rupture in 19/25 dogs, and gallbladder neoplasia in three of three dogs with these pathologies. It falsely identified biliary sludge or mucocele in eight of 28 dogs, cholecystitis/edema in three of 15 dogs, necrosis/rupture in 13/37 dogs, and gall-bladder neoplasia in 20/59 dogs that did not have these pathologies. Contrast-enhanced ultrasound correctly identified cholecystitis/edema in 42/47 dogs, but falsely identified cholecystitis/edema in three of 18 dogs. It correctly identified necrosis/rupture, benign polypoid lesions, and gallbladder neoplasia in all dogs with no false-positive results. Findings supported contrast-enhanced ultrasound as a complement to conventional ultrasonography for dogs with suspected gallbladder pathologies such as edema, necrosis, and rupture.

EVALUATION OF CONTRAST-ENHANCED ULTRASONOGRAPHY AS A METHOD FOR DETECTING GALLBLADDER NECROSIS OR RUPTURE IN DOGS.

Gall bladder necrosis and rupture are life-threatening conditions in dogs requiring surgical intervention and early diagnosis is essential. Human patients with suspected gall bladder necrosis/rupture are commonly evaluated with contrast-enhanced ultrasonography (CEUS), however this procedure has not been described in dogs with suspected gall bladder necrosis/rupture. In a prospective diagnostic cohort study, CEUS (using SonoVue contrast medium) was performed in 93 dogs with gallbladder lesions identified by abdominal conventional ultrasonography. Necrosis/rupture was identified by CEUS as a focal lack of enhancement of the gallbladder wall. Dogs with positive CEUS finding for necrosis/rupture (complete lack of regional wall enhancement) underwent immediate surgery as did dogs with other biliary disorders requiring surgery. Dogs with negative CEUS findings or those not requiring surgery were managed medically. In cases undergoing surgery, necrosis/rupture was confirmed intraoperatively (and via histopathology). Absence of necrosis/rupture was confirmed either intraoperatively (via histopathology) or was assumed to be absent by complete recovery with medical management. Forty-nine dogs underwent surgery and cholecystectomy: 24 had necrosis/rupture. CEUS was more accurate (100% sensitive and specific) in diagnosing gallbladder wall necrosis/rupture than conventional ultrasonography (75% sensitive and 81% specific) (P < 0.03). In conclusion, CEUS provides accurate characterization of gallbladder wall integrity that can impact decisions regarding clinical management, either surgical or medical.

Use of Contrast-Enhanced Ultrasound in the Differential Diagnosis of Adrenal Tumors in Dogs.

We evaluated the diagnostic accuracy of the contrast-enhanced ultrasonography (CEUS), using a second-generation microbubble contrast agent, in differentiating the different types of adrenal mass lesions in 24 dogs. At B-mode ultrasound, 9 lesions involved the right adrenal gland, 14 the left, and 1 was bilateral. Each dog received a bolus of the contrast agent into the cephalic vein, immediately followed by a 5-mL saline flush. The first contrast enhancement of each adrenal lesion was evaluated qualitatively to assess the degree of enhancement and its distribution during the wash-in and wash-out phases, as well as the presence of non-vascularized areas and specific vascular patterns. Pathological diagnoses were determined in all dogs by histopathology or by cytology. Combining enhancement degree and vascularity resulted in the best predictive model, allowing CEUS to differentiate adrenocortical adenoma (n=10), adenocarcinoma (n=7), and pheochromocytoma (n=7) with an accuracy of 91.7% (P < 0.001). Combining enhancement degree and vascularity, CEUS can discriminate malignant versus benign adrenal lesions with a sensitivity of 100.0%, a specificity of 80.0%, and an accuracy of 91.7% (P < 0.001). In conclusion, results of this study confirm that CEUS is useful for differentiating between the different types of adrenal tumors in dogs.

Leptin receptor is expressed by epidermis and skin appendages in dog.

Leptin is a polypeptide secreted by adipocytes which binds to a specific receptor (Ob-R) that is expressed in various tissues. The wide distribution of the Ob-R suggests that leptin might exert diverse biological functions, not only by regulating energy metabolism and appetite, but also by acting as a mitogen in many cell types, including keratinocytes. In this study, the presence and localization of Ob-R was investigated in the skin of the dog using RT-PCR and immunohistochemical techniques. RT-PCR revealed the presence of Ob-R m-RNA in the skin specimens collected from the dorsal region of two smooth coat breed dogs. Through immunohistochemistry performed on the skin of five dogs, the expression of the receptor was observed in the basal layer of the epidermis, in the hair follicles as well as in the apocrine sweat and sebaceous glands. No staining for Ob-R was detected in the suprabasal epidermis layers. Strong positive signals were observed in many cells of the outer root sheath of hair follicles in growing and in regressive phases. The identification of Ob-R in the above targets suggests that leptin may play a role in the regulation of cyclic renewal of the epidermis and skin appendages in dog. This study represents an important contribution to understand the complex mechanisms that are involved in the skin biology in this species.

Evidence for a dopamine intrinsic direct role in the regulation of the ovary reproductive function: in vitro study on rabbit corpora lutea.

Dopamine (DA) receptor (DR) type 1 (D1R) has been found to be expressed in luteal cells of various species, but the intrinsic role of the DA/DRs system on corpora lutea (CL) function is still unclear. Experiments were devised to characterize the expression of DR types and the presence of DA, as well as the in vitro effects of DA on hormone productions by CL in pseudopregnant rabbits. Immunoreactivity and gene expression for D1R decreased while that for D3R increased in luteal and blood vessel cells from early to late pseudopregnant stages. DA immunopositivity was evidenced only in luteal cells. The DA and D1R agonist increased in vitro release of progesterone and prostaglandin E2 (PGE2) by early CL, whereas the DA and D3R agonist decreased progesterone and increased PGF2α in vitro release by mid- and late CL. These results provide evidence that the DA/DR system exerts a dual modulatory function in the lifespan of CL: the DA/D1R is luteotropic while the DA/D3R is luteolytic. The present data shed new light on the physiological mechanisms regulating luteal activity that might improve our ability to optimize reproductive efficiency in mammal species, including humans.

Novel regulators of rabbit reproductive functions.

This is a review of original data concerning new extra- and intracellular regulators of rabbit ovarian functions. Effects of some hormones including leptin, ghrelin, oxytocin, arginine-vasotocin, endothelin (ET-1), gonadotropin releasing hormone (GnRH), adrenocorticotropic hormone (ACTH), growth factors such as insulin-like growth factor-I (IGF-I), epidermal growth factor (EGF), nuclear peroxisome proliferator-activated receptor gamma (PPARγ), pharmacological regulators of some protein kinases such as protein kinase A (PKA), mitogen-activated protein (MAP) kinase, cell division cycle protein 2 homolog (CDC2 kinase, CDK), tyrosine kinases), and plant molecules (resveratrol, rapamycin) on the functions of ovarian cells (proliferation, apoptosis, secretory activity, expression of some protein kinases) and reproductive end points (blood level of reproductive hormones, ovarian morphology, number of ovulations, embryo yield and quality, number and viability of offspring), and their possible interrelationships and practical application in rabbit breeding are reviewed.

In vivo chronic and in vitro acute effects of di(2-ethylhexyl) phthalate on pseudopregnant rabbit corpora lutea: possible involvement of peroxisome proliferator-activated receptor gamma.

The in vivo chronic and in vitro acute effects of di(2-ethylhexyl) phthalate (DEHP) on the reproductive function of peroxisome proliferator-activated receptor gamma (PPARG) were studied in rabbit corpora lutea (CL) at early stage (Day 4), midstage (Day 9), and late stage (Day 13) of pseudopregnancy. The rabbits were in vivo treated with DEHP for 15 days before induction of pseudopregnancy. Immunohistochemistry provided evidence for the presence of PPARG, prostaglandin endoperoxide synthase 1 (PTGS1), PTGS2, prostaglandin E2-9-ketoreductase (PGE2-9-K), and 3beta-hydroxysteroid dehydrogenase (3beta-HSD) in all the luteal cells during pseudopregnancy. DEHP decreased progesterone plasma levels and CL production in all the luteal stages and PPARG protein and gene expressions in early and mid-CL. DEHP in vivo treatment reduced PTGS2 protein expression at the late stage and that of PGE2-9-K at all the stages, whereas PTGS1 and 3beta-HSD were not affected. In in vitro cultured CL, DEHP alone, the PPARG antagonist T0070907 alone, or DEHP plus T0070907 diminished progesterone production and 3beta-HSD activity and increased PGF2alpha and PTGS2 in early and mid-CL, whereas DEHP plus the PPARG agonist 15d-PGJ2 did not affect these hormones and enzymes. All the in vitro treatments did not affect PGE2 secretion as well as PTGS1 and PGE2-9-K enzymatic activities in all the luteal stages. These results provided evidence that DEHP favors functional luteolysis of pseudopregnant rabbit CL, with a mechanism that seems to involve PPARG expression down-regulation, an increase of PTGS2 activity and prostaglandin F2alpha secretion, 3beta-HSD down-regulation, and decrease in progesterone.