Rabbit Corpus Luteum Research Articles

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.

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.

Farnesoid X receptor immunolocalization in reproductive tissues of adult female rabbits

Farnesoid X receptor (FXR) has been involved in lipid metabolism, cell proliferation, apoptosis, and aromatase expression, as well as in the steroid synthesis and signaling. Considering that these events occur in reproductive tissues in females, the aim of the present study was to determine the immunolocalization of FXR in the ovary, oviduct, uterus, and vagina of rabbits. Rabbits were sacrificed and their reproductive tissues were excised and histologically processed. Immunohistochemistry for FXR was done and reproductive tissues were photographed. FXR immunoreactivity was found in all types of ovarian follicles, ovarian stroma, and corpus luteum of virgin and pregnant rabbits. Also, oviductal and vaginal epithelium of virgins, as well as the oviductal smooth muscle, showed anti-FXR immunoreactivity. The uterine epithelium and musculature of virgins had scarce anti-FXR immunoreactivity. Although the role of FXR in female reproductive tissues is still not known, it is possible to consider various functions related to the reproductive tissue.

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 Gamma1

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.

Life or Death Decisions in the Corpus Luteum

The corpus luteum (CL) is an ephemeral endocrine organ. During its lifespan, it undergoes a period of extremely rapid growth that involves hypertrophy, proliferation and differentiation of the steroidogenic cells, as well as extensive angiogenesis. The growth phase is followed by a period in which remodelling of the tissue ceases, but it engages in unparalleled production of steroids, resulting in extraordinarily high metabolic activity within the tissue. It is during this stage that a critical juncture occurs. In the non-fertile cycle, uterine release of prostaglandin (PG)F(2α) initiates a cascade of events that result in rapid loss of steroidogenesis and destruction of the luteal tissue. Alternatively, if a viable embryo is present, signals are produced that result in rescue of the CL. This review article summarizes the major concepts related to the fate of the CL, with particular focus on recent insights into the mechanisms associated with the ability of PGF(2α) to bring about complete luteolysis. It has become clear that the achievement of luteolysis depends on repeated exposure to PGF(2α) and involves coordinated actions of heterogeneous cell types within the CL. Together, these components of the process bring about not only the loss in progesterone production, but also the rapid demise of the structure itself.

Expression of type I GNRH receptor and in vivo and in vitro GNRH-I effects in corpora lutea of pseudopregnant rabbits

The expression of type I GNRH receptor (GNRHR-I) and the direct role of GNRH-I on corpora lutea (CL) function were studied in the pseudopregnant rabbit model. Immunohistochemistry evidenced GNRHR-I and GNRH-I in luteal cells at early (day 4 pseudopregnancy)-, mid (day 9)-, and late (day 13)-luteal stages. Real-time RT-PCR and western blotting revealed GNRHR-I mRNA and protein at the three luteal stages. Buserelin in vivo treatment at days 9 and 13 decreased plasma progesterone levels for 48 and 24 h respectively. In in vitro cultured CL, buserelin reduced progesterone secretion, increased prostaglandin F(2α) (PGF(2α)) secretion and cyclo-oxygenase-2 (COX-2) and nitric oxide synthase (NOS) activities at days 9 and 13, and decreased PGE₂ at day 13. Co-incubation with antagonists for GNRH-I (antide), inositol 1,4,5-trisphosphate (IP₃, 2-amino-ethoxydiphenylborate), and diacylglycerol (DAG, 1-hexadecyl-2-acetyl glycerol) or inhibitors for phospholipase C (PLC, compound 48/80), and protein kinase C (PKC, staurosporine) counteracted the buserelin effects. Buserelin co-incubated with COX inhibitor (acetylsalicylic acid) increased progesterone and decreased PGF(2α) and NOS activity at days 9 and 13, whereas co-incubation with NOS inhibitor (N-nitro-l-arginine methyl ester) increased progesterone at the same luteal stages. These results suggest that GNRHR-I is constitutively expressed in rabbit CL independently of luteal stage, whereas GNRH-I down-regulates directly CL progesterone production via PGF(2α) at mid- and late-luteal stages of pseudopregnancy, utilizing its cognate type I receptor with a post-receptorial mechanism that involves PLC, IP₃, DAG, PKC, COX-2, and NOS.

A REVIEW OF LUTEOLYTIC AND LUTEOTROPHIC EFFECTS OF PROSTAGLANDINS ON THE CORPUS LUTEUM OF PSEUDOPREGNANT RABBITS : SOME IN VIVO AND IN VITRO INSIGHTS

For many years prostaglandin E2 and prostaglandin F2a have been known to exert luteotrophic and luteolytic actions on the corpus luteum (CL) of most mammalian species. Although the modalities by which the CL regulates normal reproductive cycles and maintenance of pregnancy are basically similar, increasing evidence suggests that severa! unique mechanisms regulating the growth and regression of CL exist between different species. In this respect, the corpus luteum of rabbits has not been as extensively studied as that of other farm or laboratory animals and very often the experimental data conflict. This reviews presents what is currently known about the actions of these two prostaglandins by critically examining their in vivo and in vitro effects. In non-pregnant and normally cycling animals, uterine PGF2a causes the CL to spontaneously regress at approximately 14-16 days post-ovulation depending on the species. Exogenous PGF2a administration may be used to cause CL regression and thus control the ovarian cycle. In the rabbit, however, because it does not have a defined cycle, this luteolytic mechanism comes into play only in the case of pseudopregnancy or pregnancy. The PGF2a analogue, alfaprostol, will reduce the diestrous phase at day 9 of pseudopregnancy In pregnant rabbits, PGF2a (and its synthetic analogues) is the hormone of choice for induction and synchronisation of kindling, having beneficial effects on postpartum fertility.

Intraluteal regulation of prostaglandin F2α-induced prostaglandin biosynthesis in pseudopregnant rabbits

The objective of the present study was to investigate in rabbit corpora lutea (CL), at both the cellular and molecular level, intraluteal cyclooxygenase (COX)-1, COX-2 and prostaglandin (PG) E2-9-ketoreductase (PGE2-9-K) enzymatic activities as well as in vitro PGE2 and PGF2alpha synthesis following PGF2alpha treatment at either early- (day-4) or mid-luteal (day-9) stage of pseudopregnancy. By immunohistochemistry, positive staining for COX-2 was localized in luteal and endothelial cells of stromal arteries at both the stages. In CL of both stages, basal COX-2 mRNA levels were poorly expressed, but rose (P < 0.01) 4- to 10-fold 1.5-6 h after treatment and then gradually decreased within 24 h. Compared to mid-stage, day-4 CL had lower (P < 0.01) COX-2 and PGE2-9-K basal activities, and PGF2alpha synthesis rate, but higher (P < 0.01) PGE2 production. Independent of luteal stage, PGF2alpha treatment did not affect COX-1 activity. In day-4 CL, PGF2alpha induced an increase (P < 0.01) in both COX-2 activity and PGF2alpha synthesis, whereas that of PGE2 remained unchanged. In day-9 CL, PGF2alpha up-regulated (P < 0.01) both COX-2 and PGE-9-K activities, and PGF2alpha production, but decreased (P < 0.01) PGE2 synthesis. All changes in gene expression and enzymatic activities occurred within 1.5 h after PGF2alpha challenge and were more marked in day-9 CL. Our data suggest that PGF2alpha directs intraluteal PG biosynthesis in mature CL, by affecting the CL biosynthetic machinery to increase the PGF2alpha synthesis in an auto-amplifying manner, with the activation of COX-2 and PGE-9-K; this may partly explain their differentially, age-dependent, luteolytic capacity to exogenous PGF2alpha in rabbits.

Ob receptor in rabbit ovary and leptin in vitro regulation of corpora lutea

We studied leptin involvement in rabbit corpora lutea (CL) activity, and its post-transcriptional signalling pathway. The expression of leptin receptor (Ob-R) in rabbit ovary at day 9 of pseudopregnancy was evaluated by immunohistochemistry and Western blot analysis. The specificity of the Ob-R receptor antibodies was characterised by immunoprecipitation and competition with blocking peptide. Day 9 CL were incubated in vitro with leptin alone or with inhibitors of PLC (phospholipase C), PLD (phospholipase D), AC (adenylate cyclase), JAK (janus kinase), MAPK (mitogen-activated protein kinase) and both cAMP- and cGMP-specific PDE (phosphodiesterase). Prostaglandin F2alpha(PGF2alpha), PGE2 and progesterone levels were measured in the culture medium, while NOS (nitric oxide synthase) and cAMP- and cGMP- specific PDE activities were measured in CL tissue. Positive staining for Ob-R was found within the cytoplasm of large luteal cells of CL as well as in granulosa cells of follicles and oocytes. Immunoblots detected a band of about 99 kDa size in Ob-R immunoprecipitates from CL homogenates. This band was not detectable after pre-incubation of the primary antibody with the immunising leptin peptide. Leptin increased PGF2alphaand cAMP-specific PDE, decreased basal progesterone and did not affect PGE2 and NOS levels. Leptin used the JAK pathway in increasing PGF2alpha, and MAPK and cAMP-specific PDE in decreasing progesterone. This study supports a permissive luteolytic role for leptin in rabbit CL.

Role of apoptosis in functional luteolysis in the pregnant rabbit corpus luteum: Evidence of a role for placental-derived factors in promoting luteal cell survival

Corpora lutea (CL) were isolated from one rabbit ovary on days 4, 8, 16 (peak luteal function), 28 (functional regression) and 30 of pregnancy and processed for biochemical analysis of DNA integrity. Analysis of DNA integrity revealed the presence of oligonucleosomal fragments in day 28 and day 30 CL but not in day 16 CL. The extent of low molecular weight (<15 kb) DNA labeling was 6.6 +/- 0.84 fold higher in day 30 as compared to day 16 CL (mean +/- SEM; n = 4, P < 0.01). In a second series of experiments, healthy CL collected from day 16 pregnant rabbits were incubated for 2 h in the absence or presence of 250 microg/ml of placental extract (PE) obtained from day 16 and/or day 30 placentas. Analysis of DNA integrity revealed that extensive apoptosis occurred in CL incubated in medium alone and in medium containing day 30 PE. In contrast, day 16 PE significantly suppressed apoptosis vs control (70 +/- 4%). In a third series of experiments, expression of mRNA for bcl-x and bax was measured by Northern analysis of CL treated without and with day 16 PE using cRNA probes for bcl-x and bax developed in our laboratory by RT-PCR. Treatment with PE significantly reduced bax mRNA levels but did not change bcl-x mRNA levels. These studies provide evidence that functional luteolysis in the pregnant rabbit CL is correlated with the occurrence of apoptosis. The data suggest that a factor(s) derived from the placenta may be responsible for the prolongation of CL life span during pregnancy by its ability to alter the bax:bcl -x rheostat and suppress apoptosis.

Inhibition of nitric oxide synthesis potentiates apoptosis in the rabbit corpus luteum

To determine if nitric oxide (NO) plays a role in corpus luteum (CL) physiology by affecting progesterone secretion or luteal apoptosis, an in-vitro pseudopregnant rabbit ovarian perfusion system was used to measure the effects of an inhibitor of NO synthesis, N G-nitro- l-arginine methyl ester ( l-NAME), on progesterone secretion and corpus luteal apoptosis as measured by internucleosomal DNA breakdown. Pseudopregnant rabbit ovaries perfused in vitro with l-NAME did not demonstrate any significant differences compared with control ovaries in progesterone secretion. However, apoptosis, as measured by internucleosomal breakdown, was significantly increased in l-NAME-perfused CL compared with controls. While NO does not appear to directly affect progesterone secretion, there does appear to be a role for NO in CL maintenance, or a role for inhibition of NO production in CL regression.

Regulation of nitric oxide synthase isoforms and role of nitric oxide during prostaglandin F2alpha-induced luteolysis in rabbits

Total activity of nitric oxide synthase (NOS) and the gene expression of both endothelial NOS (eNOS) and inducible NOS (iNOS) isoforms in corpora lutea of pseudopregnant rabbits were examined during prostaglandin F(2alpha) (PGF(2alpha))-induced luteolysis. Corpora lutea were collected at 0, 6, 12, 24 and 48 h after an injection of PGF(2alpha) at day 9 of pseudopregnancy. At 12 h after PGF(2alpha) administration, luteal mRNA encoding eNOS decreased (P0.05) by 40% and remained low throughout the subsequent 36 h, whereas eNOS protein increased (P0.05) two- to threefold. By contrast, expression of mRNA encoding iNOS was poor and remained fairly constant, but transcription increased eightfold (P0.01) within 6 h after PGF(2alpha) treatment and then decreased to values similar to those of controls. Total NOS activity increased twofold (P0.01) at 6 h after treatment and remained high thereafter, whereas progesterone concentrations in explanted corpora lutea decreased (P0.01) from 302.4+/-42.3 pg x mg(-1) at day 9 to 58.6+/-8.3 at 48 h later, and peripheral plasma concentrations of progesterone declined too. Long-term administration of Nomega-nitro-L-arginine methyl ester (0.6 g l(-1) per os) from day 2 of pseudopregnancy onward partially blocked the luteolytic action of PGF(2alpha) administered at day 9 of pseudopregnancy. In nitric oxide (NO)-deficient rabbits, progesterone concentrations remained higher (P0.01) than in controls at 24-48 h after PGF(2alpha) administration (4.5 to 3.2 ng x ml(-1), respectively). These data are the first to characterize NOS activity. The time course of expression of eNOS and iNOS in rabbit corpora lutea during PGF(2alpha)-induced luteolysis gives additional support to a physiological role of NO in the regulation of regression of corpora lutea in rabbits.

Regulation of nitric oxide synthase isoforms and role of nitric oxide during prostaglandin F2alpha-induced luteolysis in rabbits.

Total activity of nitric oxide synthase (NOS) and the gene expression of both endothelial NOS (eNOS) and inducible NOS (iNOS) isoforms in corpora lutea of pseudopregnant rabbits were examined during prostaglandin F(2alpha) (PGF(2alpha))-induced luteolysis. Corpora lutea were collected at 0, 6, 12, 24 and 48 h after an injection of PGF(2alpha) at day 9 of pseudopregnancy. At 12 h after PGF(2alpha) administration, luteal mRNA encoding eNOS decreased (P<or=0.05) by 40% and remained low throughout the subsequent 36 h, whereas eNOS protein increased (P<or=0.05) two- to threefold. By contrast, expression of mRNA encoding iNOS was poor and remained fairly constant, but transcription increased eightfold (P<or=0.01) within 6 h after PGF(2alpha) treatment and then decreased to values similar to those of controls. Total NOS activity increased twofold (P<or=0.01) at 6 h after treatment and remained high thereafter, whereas progesterone concentrations in explanted corpora lutea decreased (P<or=0.01) from 302.4+/-42.3 pg x mg(-1) at day 9 to 58.6+/-8.3 at 48 h later, and peripheral plasma concentrations of progesterone declined too. Long-term administration of Nomega-nitro-L-arginine methyl ester (0.6 g l(-1) per os) from day 2 of pseudopregnancy onward partially blocked the luteolytic action of PGF(2alpha) administered at day 9 of pseudopregnancy. In nitric oxide (NO)-deficient rabbits, progesterone concentrations remained higher (P<or=0.01) than in controls at 24-48 h after PGF(2alpha) administration (4.5 to 3.2 ng x ml(-1), respectively). These data are the first to characterize NOS activity. The time course of expression of eNOS and iNOS in rabbit corpora lutea during PGF(2alpha)-induced luteolysis gives additional support to a physiological role of NO in the regulation of regression of corpora lutea in rabbits.

Functional and structural regression of the rabbit corpus luteum is associated with altered luteal immune cell phenotypes and cytokine expression patterns

Following attenuation of progesterone production corpora lutea are selectively cleared, a process associated with recruitment of macrophages. In the rabbit little is known about luteal immune cell phenotypes and expression of cytokines, which influence immune cells and resident luteal cells, during luteolysis. Consequently, we studied luteal immune cells by immunohistochemistry as well as luteal IL-10, TNFalpha, MCP-1, IFN-gamma, and IL-1beta mRNA expression by semiquantitative RT-PCR from day 8 to day 20 in pseudopregnant rabbits (d8-d20 p.hCG). Luteal function was assayed by serum progesterone levels. Functional luteolysis commenced by d14 p.hCG as indicated by attenuation of serum progesterone levels. X4(+) tissue macrophage levels increased transiently on d12 and d14 p.hCG, whereas CD5(+) T-cell levels transiently declined on these two days. CD68(+) macrophages increased progressively after d16 p.hCG. The luteal mRNA level of the anti-inflammatory cytokine IL-10 as well as the mRNA levels of the pro-inflammatory cytokines TNFalpha and MCP-1 increased after d16 p.hCG and remained elevated up to d20 p.hCG. IFN-gamma and IL-1beta mRNA expression did not vary systematically. In summary, luteolysis was associated with an initial transient increase of X4(+) macrophages and decrease of CD5(+) T-cells, and later recruitment of CD68(+) macrophages. During structural regression pro- and anti-inflammatory cytokines are upregulated possibly to control immune cell function.

Expression patterns of endothelial and inducible nitric oxide synthase isoforms in corpora lutea of pseudopregnant rabbits at different luteal stages.

Total activity of nitric oxide (NO) synthase (NOS) and expression of both endothelial (eNOS) and inducible (iNOS) isoforms were examined in corpora lutea (CL) of rabbits across pseudopregnancy by quantitative RT-PCR analysis, Western blot and immunohistochemistry. CL were collected at early- (day 4), mid- (day 9) and late- (day 13) luteal phases of pseudopregnancy. The PCR product of rabbit luteal eNOS was cloned and its direct sequence exhibited 90% homology with those of other species. The steady-state mRNA levels encoding eNOS remained fairly constant throughout both early- and mid-luteal stages of pseudopregnancy but dropped almost to half (P</=0.05) by day 13. By contrast, luteal eNOS proteins increased 2-fold (P</=0.05) from the early- to late-luteal phase. Independently of CL age, iNOS mRNA was very poorly expressed while protein levels gradually declined from the early- to late-luteal stage. Intense eNOS-like immunoreactivity was detected in large luteal cells, while iNOS staining was targeted to a few, isolated cells, probably macrophages. Basal NOS activity was greater in day 4 CL than in both day 9 and day 13 CL. These data are the first to characterize in rabbit CL the temporal expression patterns of NOS isoforms across different luteal stages of pseudopregnancy and, collectively, suggest the existence of an expressional control for this constitutive isoform, which might have a physiological role in regulating CL function during development.

Prostaglandin receptors and role of G protein-activated pathways on corpora lutea of pseudopregnant rabbit in vitro.

Studies were conducted to characterize receptors for prostaglandin (PG) F(2alpha) (PGF(2alpha)) and PGE(2), and the signalling pathways regulating total nitric oxide synthase activity and progesterone production in rabbit corpora lutea (CL) of different luteal stages. CL were obtained at days 4, 9 and 13 of pseudopregnancy and cultured in vitro for 2 h with PGF(2alpha) or PGE(2) and with activators and inhibitors of G protein (Gp), phospholipase C (PLC), protein kinase C (PKC), adenylate cyclase (AC) and protein kinase A (PKA). High affinity PGF(2alpha) receptor (K(d)=1.9+/-0.6 nM mean+/-s.e.m. ) concentrations increased (P< or =0.01) four- to five-fold from early to mid- and late-luteal phases (50.6+/-8.5, 188.3+/-36.1 and 231.4+/-38.8 fmol/mg protein respectively). By contrast, PGE(2) receptor (K(d)=1.6+/-0.5 nM) concentrations decreased (P< or =0.01) from day 4 to day 9 and 13 (27.5+/-7.7, 12.4+/-2.4 and 16.5+/-3.0 fmol/mg protein respectively). The Gp-dependent AC/PKA pathway was triggered only on day 4 CL, mimicking the PGE(2) treatment and increasing progesterone production. In both day 9 and day 13 CL, the Gp-activated PLC/PKC pathway evoked a luteolytic effect similar to that induced by PGF(2alpha). The time-dependent selective resistance to PGF(2alpha) and PGE(2) by rabbit CL is mediated by factors other than a lack of luteal receptor-ligand interactions.

Inhibitors of caspase homologues suppress an apoptotic phenotype in cultured rabbit corpora lutea

Apoptosis is a morphologically defined type of cell death initiated by various stimuli that results in the activation of caspases (cysteine-containing aspartate-specific proteases). In the present study, it was determined that caspases are present during, and play a role in, corpus luteum (CL) apoptosis in vitro. Pseudopregnancy was induced in rabbits with 100 IU human chorionic gonadotrophin. On Day 11 of pseudopregnancy, CL were isolated and cultured for 0, 2, 4, 6, and 8 h in the absence of trophic support to induce spontaneous apoptosis. Total RNA was extracted and analysed for caspase-I expression by Northern blot analysis. The results demonstrated caspase-I expression from 4 h. In the second part of the study, CL were incubated without trophic support for 4 h with increasing concentrations of three general caspase inhibitors, sodium aurothiomalate (SAM), iodoacetic acid (IAA) and N-tosyl-L-phenylalanylchloromethylketone (TPCK), and two specific caspase inhibitors, N-acetyl (Ac)-Tyr-Val-Ala-Asp (YVAD)-chloromethylketone (CMK) (Ac-YVAD-CMK) and Ac-Asp-Glu-Val-Asp (DEVD)-aldehyde (CHO) (Ac-DEVD-CHO). At completion, DNA was isolated and integrity assessed. Treatment of CL with SAM, IAA or Ac-DEVD-CHO effectively suppressed apoptotic DNA fragmentation. The final component of the study was to examine caspase-3 protein expression. Western blot analysis revealed a significant increase in caspase-3 expression over the experimental time-course. The results of the present study clearly demonstrate a time-dependent link between the caspases, specifically caspase-3 and spontaneous apoptosis in the rabbit CL.

Nitric oxide synthase activity and progesterone release by isolated corpora lutea of rabbits in the early and mid-luteal phases of pseudopregnancy are modulated differently by prostaglandin E-2 and prostaglandin F-2alpha via adenylate cyclase and phospholipase C.

By examining in vitro the effects of prostaglandin E-2 (PGE-2) and prostaglandin F-2alpha (PGF-2alpha) induced in the corpora lutea (CL) of pseudopregnant rabbits, we have demonstrated that these prostaglandins modulate luteal nitric oxide synthase (NOS) activity and progesterone production differently, depending on the age of the CL. On CL obtained on day 4 of pseudopregnancy (day-4), PGE-2 was found to depress NOS total activity to 13% of control and to significantly increase basal progesterone secretion by 61%, while PGF-2alpha had no effect. On day-9 CL, PGE-2 was ineffective, but PGF-2alpha caused a 2.5-fold increase of NOS activity and a marked decrease in progesterone production. Using specific inhibitors, we found that the regulatory actions of PGE-2 in vitro are mediated via the adenyl cyclase/protein kinase A (PKA) second messenger system, while the PGF-2alpha-induced luteolytic effects on day-9 CL depend upon activation of the phospholipase C/protein kinase C (PKC) system. The different responsiveness of day-4 and day-9 CL to PGE-2 and PGF-2alpha could depend on receptor availability for these two prostaglandins, even if other cellular mechanisms cannot be excluded. The present study supports a functional role for NOS in regulating the steroidogenic capacity of rabbit CL, and reveals a novel interaction between a stimulatory G-protein-coupled receptor and PKC/PKA-mediated signal transduction modulating NOS activity.

Antioxidants Mimic the Ability of Chorionic Gonadotropin to Suppress Apoptosis in the Rabbit Corpus Luteum in Vitro: A Novel Role for Superoxide Dismutase in Regulating bax Expression

Antioxidants Mimic the Ability of Chorionic Gonadotropin to Suppress Apoptosis in the Rabbit Corpus Luteum in Vitro: A Novel Role for Superoxide Dismutase in Regulating bax Expression

Antioxidants mimic the ability of chorionic gonadotropin to suppress apoptosis in the rabbit corpus luteum in vitro: a novel role for superoxide dismutase in regulating bax expression.

We have recently reported that members of the bcl-2 gene family are expressed and estradiol regulated in rabbit luteal cells during corpus luteum (CL) regression, and that estradiol and hCG are effective inhibitors of apoptosis in the rabbit CL in vivo and in vitro. As Bcl-2 and related proteins are known to regulate levels of reactive oxygen species or their intermediates in cells as one possible mechanism to control apoptosis, the present studies were designed to examine if oxidative stress plays a role in luteal cell apoptosis during CL regression in the rabbit. In the first set of experiments, healthy CL obtained from day 11 pseudopregnant rabbits were incubated in serum-free medium for 2 h in the absence or presence of superoxide dismutase (SOD; 1.5-150 U/ml), ascorbic acid (1-100 mM), N-acetyl-L-cysteine (25 and 50 mM), or catalase (10-1000 U/ml). Cells within CL incubated in medium alone exhibited extensive apoptosis (examined by analysis of extracted DNA using 3'-end labeling), and this onset of apoptosis was blocked in a dose-dependent fashion by treatment with SOD, ascorbic acid, N-acetyl-L-cysteine, or catalase. In the second set of experiments, expression of bax and bcl-x in CL after in vitro treatment without and with 100 U/ml SOD was examined. Although SOD treatment did not alter the levels of bcl-x messenger RNA (mRNA) over the 2-h incubation period, this antioxidant enzyme significantly reduced the levels of bax mRNA in incubated CL. In the final set of experiments, we observed that expression of mitochondrial- or manganese-containing SOD was significantly increased by treatment of isolated CL with 1 microg/ml hCG in vitro, whereas bax mRNA levels were significantly reduced under the same culture conditions. Collectively, these data indicate that the gonadotropin-mediated inhibition of apoptosis in rabbit luteal cells involves enhanced expression of the oxidative stress response gene, manganese-containing SOD, whose protein product may then function to protect luteal cells directly from the damaging effect of reactive oxygen species and/or indirectly by acutely down-regulating expression of Bax, a prooxidant member of the Bcl-2 protein family.