Corpora Lutea Of Pseudopregnant Rabbits 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.

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.

Expression patterns of cytokines, p53 and nitric oxide synthase isoenzymes in corpora lutea of pseudopregnant rabbits during spontaneous luteolysis.

The gene expressions for macrophage chemoattractant protein-1 (MCP-1), interleukin (IL)-1 beta, IL-2 and p53 were examined by semi-quantitative RT-PCR in corpora lutea (CL) of rabbits during spontaneous luteolysis at days 13, 15, 18 and 22 of pseudopregnancy. In the same luteal tissue, total activity of nitric oxide (NO) synthase (NOS) and genes for both endothelial (eNOS) and inducible (iNOS) isoforms were also analysed. From day 13 to 15, MCP-1 and IL-1 beta mRNA levels rose (P < or = 0.01) almost 2-fold, and the transcript for p53 almost 8-fold, but then all dropped (P < or = 0.05) from day 18 onward. IL-2 mRNA abundance was higher (P < or = 0.01) on day 13 and then gradually declined. During luteolysis, eNOS mRNA decreased 40% (P < or = 0.05) by day 15, but thereafter remained unchanged, while iNOS mRNA was barely detectable and did not show any clear age-related pattern throughout the late luteal stages. Total NOS activity progressively increased (P < or = 0.01) from day 13 to 18 of pseudopregnancy and then dropped to the lowest (P < or = 0.01) levels on day 22. Luteal progesterone content also declined during CL regression from 411 to 17 pg/mg found on days 13 and 22 respectively, in parallel with the decrease in blood progesterone concentrations. These data further support a physiological role of NO as modulator of luteal demise in rabbits. Locally, luteal cytokines may be involved in the up-regulation of NOS activity, while downstream NO may inhibit steroroidogenesis and induce expression of p53 gene after removal of the protective action of progesterone.

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.

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.

An immunocytochemical study of the progesterone receptor in rabbit ovary

Distribution of progesterone receptor (PR) was studied immunohistochemically in rabbit ovary and uterus using a monoclonal anti-receptor antibody. At the subcellular level, PR was located in cell nuclei of prepubertal rabbits, which were either non-treated, primed with estrogen or made pseudopregnant. At the tissue level, germinal epithelium, the external theca cell layer and granulosa cell layer were PR-positive. The internal theca cell layer had no PR immunoreactivity. The corpora lutea of pseudopregnant rabbits contained small amounts of PR in some of the animals. During pseudopregnancy, the distribution and staining intensity of PR-positive cells in the ovary was essentially similar to that in non-treated rabbits except for weak PR immunoreactivity in the internal theca cell layer. This differed significantly from the uterus, in which pseudopregnancy caused a marked decrease in PR immunoreactivity. This implies that receptor downregulation by endogenous progesterone is not the same in different organs and cell types. Immunohistochemical techniques give valuable information as to the steroid hormone target cell types and their distribution which are not available by conventional steroid-binding assays.

Estradiol-induced changes in rabbit luteal cell progestin production and cholesterol and cholesterol ester content

Estradiol deprivation in vivo causes accumulation of cholesterol and cholesterol ester in the corpora lutea of pseudo-pregnant rabbits within 24 hours. These accumulations occur concomitantly with an abrupt cessation of progestin production. Retreatment with estradiol restores progestin production and can block further increases in cholesterol accumulation (per mg total tissue protein). Ample quantities of precursor cholesterol ester and cholesterol substrate are present in corpora lutea when progestin falls because of estradiol deprivation. We conclude therefore that the gonadotropic action of estradiol is, in part, because of its stimulation of cholesterol conversion to progestins.

Monoclonal antibody recognition of estrogen receptor in rabbit corpus luteum

We demonstrated previously by administering [3H]estradiol and using autoradiography that, in vivo, the exogenous estradiol that will sustain progesterone production is sequestered to nuclei of the luteal cells in corpora lutea of pseudopregnant rabbits. Our objective in the present experiments was to use an immunological method to demonstrate that estrogen receptor of the rabbit corpus luteum is associated with this uptake. For this purpose, we prepared nuclear extracts from corpora lutea removed from anesthetized pseudopregnant rabbits 10 min after arterial infusion of 125I-estradiol. These extracts were then analyzed by sucrose density gradient centrifugation in the presence and absence of monoclonal antibody against human breast cancer estrogen receptor. We found that the 125I-estradiol-binding component extracted with 0.5 M KCl from the nuclear pellet sediments in the 3-4S region in high-salt gradients; in the presence of monoclonal antibody (D-547 Sp2 gamma), the entire estradiol-binding component is shifted to the 8-9S region, indicating the formation of immunocomplex with the 125I-estradiol-receptor moiety. The principal estradiol-binding component that can be observed in the 3-4S region of high-salt gradients of cytosol of rabbit corpora lutea reacts with this antibody regardless of whether 125I- or [3H]estradiol is given in vivo or in vitro. In both cytosol and nuclear extracts of luteal tissue, unlabeled diethylstibestrol totally inhibits the binding of radiolabeled estrogen to this antibody-shifted 125I- or [3H]estradiol-binding moiety; neither progesterone nor testosterone inhibit binding of radiolabeled estrogen to the antibody-shifted moiety. From these findings we conclude that sequestering of estradiol by the rabbit corpus luteum in vivo is accomplished by the steroid-specific estrogen receptor that shares characteristics of the estrogen receptor found in human breast cancers, including a) antigenic features and b) the capacity to translocate estrogens from the cytosol to the nuclear fraction.

Cell contacts in rabbit corpora lutea

The cell contacts in corpora lutea of pseudopregnant rabbits were studied in normally processed and in lanthanum treated tissue. Gap junctions and septatelike cell contacts were detected and are described in this paper. The gap junctions are pentalaminar or show a 20 Å gap after uranyl staining “en bloc.” Tangentially cut gap junctions in lanthanum treated tissue show the generally known tightly packed intercellular particles. In septatelike cell contacts the cell membranes are separated from each other by a 160–180 Å wide intercellular space and the contact is made by repeating electron lucent septa. After colloidal lanthanum application the cross-sectioned cell contacts also show repeating structures with a periodicity ranging from 80–95 Å. These structures seem to be composed of a larger particle in the middle and symmetrically arranged smaller particles at both sides near the cell membrane. The “en face” view shows clear granules in a dark background. These granules and also their distribution are, however, clearly different from the particles seen in gap junctions. These findings are compared with earlier results on septatelike cell contacts and the presumed function of these septatelike cell contacts is discussed.

Effect of hysterectomy on luteinizing hormone-induced Corpora lutea of pseudopregnant rabbits.

Effect of hysterectomy on luteinizing hormone-induced Corpora lutea of pseudopregnant rabbits.