Pseudopregnant Rabbits Research Articles

Transplantation of Aceh cattle ovary into the uterus of pseudopregnant local rabbits: Effect of post-transplant stress on uterine histopathology and ovarian follicle dynamics.

The increase in the levels of the cortisol hormone caused by the stress conditions generated by an ovary transplantation procedure can damage the uterus of the transplant recipient as well as the transplanted ovaries. This study aimed to analyze the histopathological changes that occur in the uterine horn of pseudopregnant local rabbits (recipients), as well as the ovarian follicular integrity of the donor Aceh cattle after transplantation. After 30 days of adaptation, all rabbits were divided into three treatment groups: R1 (the group of rabbits that underwent ovarian transplantation for 3 days, n = 5), R2 (the group of rabbits that underwent ovarian transplantation for 5 days, n = 5), and R3 (the group of rabbits that underwent ovarian transplantation for 7 days, n = 5). Pseudopregnancy induction was performed using the pregnant mare's serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) methods. The rabbits were injected with 100 IU of PMSG intramuscularly, followed by an injection of 75 IU of hCG intravenously 3 days later. Ovarian transplantation was performed on day 8 (day 0 was the day of hCG injection). The concentration of cortisol hormone metabolites was measured from fecal samples using an enzyme-linked immunosorbent assay technique. The uterus and ovaries were collected for histopathological and follicular dynamics examination after the transplantation process was completed. The mean cortisol levels (ng/g) recorded before versus after the transplant in the R1, R2, and R3 groups were 146.23 ± 17.60 versus 338.84 ± 302.79, 128.97 ± 81.56 versus 174.79 ± 101.70, and 124.88 ± 43.61 versus 321.91 ± 221.63 (p < 0.05), respectively. The examination of the histopathological appearance of the uterus revealed edema in the uterine lumen, hyperemia and hemorrhage in the endometrium, necrosis of the epithelium, and infiltration of inflammatory cells. Hemorrhage and hyperemia were severe and filled the endometrium in the R1 compared with the R2 and R3 animals. Ovarian follicle development occurred in all treatment groups, although some histopathological features were observed. The number of tertiary follicles in R1, R2, and R3 animals was 24.67 ± 7.37, 20.67 ± 7.57, and 9.67 ± 3.79 (p < 0.05), respectively. Based on the results of this study, it can be concluded that the transplantation of ovaries from Aceh cattle into pseudopregnant local rabbits triggered an increase in the levels of the cortisol hormone and uterine histological changes; however, follicles were still detected at various stages of development in the transplanted Aceh cattle ovaries. The results of this study are valuable for clinicians and researchers because they provide information regarding an alternative in vivo ovarian preservation technique using pseudopregnant rabbits.

Evaluation of Estrogen Alpha Receptor (ER-Α) and Progesterone-A Receptor (PR-A) Expression in the Oviducts and Uterus of Indonesian Local Rabbits after Induced by Several Method of Pseudo-Pregnancy

The objective of this study was to determine the expression of ER-A and PR-A in the oviduct and uterus of pseudo-pregnant rabbits after induced by several induction methods of pseudo-pregnancy. This study used 30 local rabbits, aged 1 - 1.5 years, body weight of 2.8 - 3.0 kg and clinically healthy. Rabbits were divided into 6 groups (n = 5), namely: K1 (negative control, given 1.5 mL physiological NaCl injection), K2 (positive control, given 100 IU eCG injection and mated with male rabbits, followed by 75 IU hCG injection 3 days later), K3 (given 100 IU eCG injection followed by injection of 75 IU hCG 3 days later), K4 (artificial copulation was performed by inserting a 1 cm long cotton bud into the rabbit’s vagina), K5 (ovulation induction by injection of 50 IU hCG) and K6 (ovulation induction by injection of 5 g GnRH). On the 8th day after treatment, rabbits were euthanized and their oviduct and uterus organs were taken for histological preparation and stained using the immunohistochemical staining. The expression of ER-A and PR-A was valued using the intensity score and analyzed using the Kruskal-Wallis test followed the Mann Whitney U test. The expression ER-A in the infundibulum, ampullary and isthmus of K1 was significantly different (p &lt; 0.05) from K2 - K6 rabbits. The ER-A expression in the endometrium of uterine horn of K1 and K3 was significantly different with K2, K4, K5 and K6 rabbits (p &lt; 0.05). Furthermore, PR-A expression in oviducts of K1 and K3 was lower than that in K2, K3, K5 and K6 (p &lt; 0.05) and the expression in the endometrium, uterine glands and myometrium of K2 - K6 was higher than K1 rabbits (p &lt; 0.05). In conclusion, the hormonal induction could be used to induce pseudopregnancy in local rabbits based on the ER-A and PR-A expressions in oviduct and uterus. HIGHLIGHTS Pseudo-pregnant rabbits are needed for research purposes in the field of reproduction Several methods of induction of pseudo-pregnancy in rabbits have been carried out in this study Pseudo-pregnancy induction cause changes in the concentration of reproductive hormones (progesterone and estrogen) These changes can be detected based on the presence of receptors for these two hormones (PRA and ERα) in reproductive tract of four groups of rabbits and the results were then evaluated GRAPHICAL ABSTRACT

Gene Targeting in Rabbits: Single-Step Generation of Knockout Rabbits by Microinjection of CRISPR/Cas9 Plasmids.

The development of genome editing technology has allowed gene disruptions to be achieved in various animal species and has been beneficial to many mammals. Gene disruption using pluripotent stem cells is difficult to achieve in rabbits, but thanks to advances in genome editing technology, a number of gene disruptions have been conducted. This chapter describes a simple and easy method for carrying out gene disruptions in rabbits using CRISPR/Cas9 in which the gene to be disrupted is marked, the presence or absence of off-target candidates is checked, and a plasmid allowing simultaneous expression of Cas9 and sgRNA is constructed. Next, the cleaving activity of candidate sequences is investigated, and assessments are carried out to determine whether the target sequences can be cut. Female rabbits subjected to superovulation treatment are mated with male rabbits and fertilized eggs are collected, and then pronuclear injection of plasmid DNA is performed. The next day, the two-cell stage embryos are transplanted into a pseudopregnant rabbits, and offspring are born within approximately 29-30days. The genomic DNA of the offspring is then examined to check what type of genetic modifications has occurred. With the advent of CRISPR/Cas9, the accessibility of gene disruptions in rabbits has improved remarkably. This chapter summarizes specifically how to carry out gene disruptions in rabbits.

Evaluation of infrared thermography findings in pseudopregnant rabbit

The effectiveness of infrared thermography (IRT) in the diagnosis of pseudopregnant rabbits, the change of these images in the pseudopregnancy process, and its relationship with serum progesterone (P4) level was investigated. Fourteen healthy female rabbits of New Zealand breed were divided into two study groups. Pseudopregnancy induction was performed in the first study group with GnRH injection (0.2 ml Buserelin acetate, intramuscularly) on the 0th day of the study (Group 1, n=7). The second group (Group 2, n=7) was given placebo (0.2 ml 0.9% NaCl, intramuscularly). Rectal temperature was taken from all rabbits on days 0-5-10 and 15, eye, nasal tip, and vulvar IRT were applied, and serum P4 valueswere measured by Enzyme-linked immunosorbent assay (ELISA) method. In the pseudopregnant group, the temperatures of the eyes, nose, and vulva were higher on the 10th day (p

Comparison of four methods of inducing pseudopregnancy in rabbits

This study was conducted to compare several methods for inducing pseudopregnancy in local rabbits. The study used 25 clinically healthy rabbits of local breeds, aged 1-1.5 years, with a bodyweight of 1.8-2.2 kg. The rabbits were divided into five treatment groups (n = 5): R1, R2, R3, R4, and R5. Each group was given a different treatment. Group R1 was injected intramuscularly with 100 IU PMSG and mated with males three days later, which was followed by an intravenous injection of 75 IU hCG. Pseudopregnancy in group R2 was induced through artificial copulation by inserting a 1 cm long cotton bud into vaginal lumen at 5 a.m. Pseudopregnancy in group R3 was induced using 50 IU hCG intramuscularly. Group R4 was injected intravenously with 5 g GnRH and then intramuscularly with 100 IU PMSG and three days later with 75 IU hCG. Blood samples were collected on days 2, 4, 6, and 8 for estrogen and progesterone measurement, whereas an ovarian histological observation of all groups was performed on day 8. The results show that the highest concentration of estrogen was obtained in group R2, which was significantly different from the other groups (P &lt;0.05), but the difference in examination time was not statistically significant (P &gt; 0.05). Furthermore, the concentration of progesterone in all groups fluctuated at various times of the day. Progesterone concentrations were higher in group R5, in which pseudopregnancy was induced with PMSG and hCG. Group R5 showed a significant difference (P &lt;0.05) with R2, R3, and R4, but the difference with the positive control group (R1) was not significant (P &gt; 0.05). The numbers of corpora lutea in groups R1, R2, R3, R4, and R5 were, respectively, 6.87 ± 1.58, 6.22 ± 2.71, 6.74 ± 1.94, 5.98 ± 2.04, and 8.8 ± 1.90, and the largest diameter was obtained in R5 (1.65 ± 0.37 mm). In conclusion, the best method of inducing pseudopregnancy in local rabbits is the administration of PMSG and hCG.

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

Simple SummaryCorpora lutea (CL) are temporary endocrine structures that secrete progesterone, which is essential for maintaining a healthy pregnancy. A variety of regulatory factors come into play in modulating the functional lifespan of CL, with luteotropic and luteolytic effects. Many aspects of luteal phase physiology have been clarified, yet many others have not yet been determined, including the molecular and/or cellular mechanisms that maintain the CL from the beginning of luteolysis during early CL development. This paper summarizes our current knowledge of the endocrine and cellular mechanisms involved in multifactorial CL lifespan regulation, using the pseudopregnant 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.

Energy homeostasis in rabbit does during pregnancy and pseudopregnancy

This study was conducted to evaluate the changing concentrations of metabolic hormones and metabolites in pregnant (P) and pseudopregnant (PP) rabbit does. Twenty-five New Zealand White rabbit does were submitted to artificial insemination (AI) and then classified as P (n = 15) or PP (n = 10). Blood samples were collected weekly until day 32 post AI. During pregnancy, leptin concentrations were greater on Days 14 and 21 (P < 0.05), while insulin was greater on days 21 and 32 post AI (P < 0.05) compared to PP does. The triiodothyronine/thyroxine (T3/T4) ratio was greater in the first and last week (P < 0.001); whereas, cortisol concentrations were greater in the last week of pregnancy and after parturition (P < 0.01) compared with that of PP does. Non-esterified fatty acids (NEFA) concentrations increased from day 7 until day 32 post AI (P < 0.05). Glucose concentrations were unchanged throughout pregnancy although concentrations were positively associated with litter size. These results indicate concentrations of hormones and metabolites change during pregnancy to ensure energy requirements are met for both the foetuses and the maternal tissues. Physiological hyperleptinemia, hyperinsulinemia, and changes in cortisol as well as thyroid hormones indicate there is an adaptation of metabolic functions induced by pregnancy. These adaptations could be mediated by gonadal steroids because changes mainly occur in the second half of pregnancy when the profile of the sex hormones differs between P and PP does.

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.

Immunohistochemical localization of progesterone receptors alpha (PRA) in ovary of the pseudopregnant rabbit.

Progesterone plays an important role in the reproductive function and follicular development in mammals. The aim of the present study was to examine the localization of progesterone receptor alpha (PRA) in ovary of pseudopregnant rabbit by immunohistochemical methods. Samples were collected from 14 h. to 18 days of pseudopregnancy. At the first stage of pseudopregnancy (14 h.), the rabbit ovary showed moderate immunostaining of PRA in the granulosa cells and theca interna cells of preovulatory follicle and in the stroma cells. At the middle stage of pseudopregnancy (3-7 days), the rabbit ovary showed strong immunostaining of PRA in ovarian surface epithelial cells, follicular cells of the primary follicle, granulosa cells and theca interna cells of the growing and antral follicles. Moderate immunoexpression of PRA were observed in the large lutein cells and endothelial cells of the corpus haemorrhagicum and corpus luteum and in the stroma cells. At the end of pseudopregnancy (18 days) strong PRA reactions were detected in the small lutein cells of the regressed corpus luteum. Moderate to strong PRA immuno-expression were observed in the proliferated theca interna cells of the atretic antral follicles. The atretic large lutein cells of the regressed corpus luteum showed negative immunostaining for PRA. This study showed that the PRA positive small lutein cells of the regressed corpus luteum and the PRA positive proliferated theca interna cells of the atretic antral follicles were transformed into PRA positive interstitial gland cells. In conclusion, the present study had described the distribution of PRA in the ovary of pseudopregnant rabbit, which is not discussed before in the available literature. It also gives more information about follicular dynamic, formation and origin of interstitial glands, mechanism of ovulation, formation and regression of the corpus luteum.

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- (4days) and mid-luteal (9days) 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 40min that was followed 50min 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 6hr later to reach basal values after 24hr. 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.

Gene Targeting in Rabbits: Single-Step Generation of Knock-out Rabbits by Microinjection of CRISPR/Cas9 Plasmids.

The development of genome editing technology has allowed gene disruptions to be achieved in various animal species and has been beneficial to many mammals. Gene disruption using pluripotent stem cells is difficult to achieve in rabbits, but thanks to advances in genome editing technology, a number of gene disruptions have been conducted. This paper describes a simple and easy method for carrying out gene disruptions in rabbits using CRISPR/Cas9 in which the gene to be disrupted is marked, the presence or absence of off-target candidates is checked, and a plasmid allowing simultaneous expression of Cas9 and sgRNA is constructed. Next, the cleaving activity of candidate sequences is investigated, and assessments are carried out to determine whether the target sequences can be cut. Female rabbits subjected to superovulation treatment are mated with male rabbits and fertilized eggs are collected, and then pronuclear injection of plasmid DNA is performed. The next day, the two-cell stage embryos are transplanted into pseudopregnant rabbits, and offspring are born within approximately 29-30days. The genomic DNA of the offspring is then examined to check what types of genetic modifications have occurred. With the advent of CRISPR/Cas9, the accessibility of gene disruptions in rabbits has improved remarkably. This paper summarizes specifically how to carry out gene disruptions in rabbits.

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.

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.

Evidence for a Luteotropic Role of Peroxisome Proliferator-Activated Receptor Gamma: Expression and In Vitro Effects on Enzymatic and Hormonal Activities in Corpora Lutea of Pseudopregnant Rabbits1

The expression of peroxisome proliferator-activated receptor gamma (PPARgamma) and its role in corpora lutea (CL) function were studied in pseudopregnant rabbits. Corpora lutea were collected at an early stage (Day 4), midstage (Day 9), and late stage (Day 13) of pseudopregnancy. Immunohistochemistry found evidence for the presence of PPARgamma in the perinuclear cytoplasm and nucleus of all the luteal cells; immunoreactivity decreased from the early to the late stage, with immunonegativity of the nuclei of late stage CL. PPARgamma mRNA transcript was expressed in all the luteal stages with the lowest level in the late stage. In CL cultured in vitro, the PPARgamma agonist (15-deoxy delta12,14 prostaglandin J2 [15d-PGJ2], 200 nM) increased and the antagonist (T0070907, 50 nM) decreased progesterone secretion at early and midluteal stages, whereas 15d-PGJ2 reduced and T0070907 increased PGF2alpha at the same stages. Prostaglandin-endoperoxide synthase 2 (PTGS2) activity was reduced by 15d-PGJ2 and increased by T0070907 in CL of early and midluteal stages. Conversely, 15d-PGJ2 increased and T0070907 reduced 3beta-hydroxysteroid dehydrogenase (3beta-HSD) activity in early and midluteal stage CL. PGE2 in vitro secretion as well as PTGS1 and 20alpha-HSD enzymatic activities were not affected by 15d-PGJ2 and T0070907 in any CL types. These results indicate that PPARgamma plays a luteotropic role in pseudopregnant rabbits, through PTGS2 down-regulation and 3beta-HSD up-regulation, with a consequent PGF2alpha decrease and progesterone increase.

Aglepristone (RU534) effects on luteal function of pseudopregnant rabbits: Steroid receptors, enzymatic activities, and hormone productions in corpus luteum and uterus

The study was designed to examine the aglepristone (RU534) mechanisms affecting the corpora lutea (CL) lifespan in pseudopregnant rabbits. Aglepristone (10mg/kg b.w.) was injected subcutaneously twice at either early- or mid-luteal phase (Days 3 and 4, or Days 8 and 9, respectively) after induction of ovulation with GnRH (Day 0). Corpora lutea and uteri, explanted at days 6 and 11, were evaluated for immunohistochemistry and Western blotting of progesterone (PR) and estrogen (ER) receptors, cyclooxygenase 1 (COX1), COX2, and PGE2-9-ketoreductase (PGE2-9-K) enzymatic activities, and progesterone, PGF2α, and PGE2 in vitro synthesis. Independent of luteal stage, aglepristone prolonged the functional luteal phase by 3 Days over that of controls as assessed by blood progesterone profiles. Aglepristone decreased protein for ER during both luteal-stages in CL and uteri. Progesterone receptor protein was decreased by RU354 at Days 6 in the uterus and at Days 11 in CL, whereas RU534 increased PR at Days 11 in uteri. In the CL, RU534 enhanced progesterone production at Days 6 and 11, whereas it decreased PGF2α and increased PGE2 at Day 11. In the uteri, RU534 decreased PGF2α and increased PGE2 synthesis at both days. COX2 and PGE2-9K activities were decreased by RU534 in the CL at Day 11, whereas in the uteri COX2 increased and PGE2-9-K decreased at Days 6 and 11. In conclusion, these data on aglepristone effects suggest that progesterone has a regulatory role on luteal function through direct and uterine-mediated mechanisms in pseudopregnant rabbits.

Direct actions of ACTH on ovarian function of pseudopregnant rabbits

The present study sought to assess whether the receptors for adrenocorticotropic hormone (ACTH), MC2R, and for glucocorticoid (GR) are expressed in corpora lutea (CL) of pseudopregnant rabbits and whether ACTH and cortisol exert any direct action on luteal function. By immunohistochemistry, positive reaction for MC2R and GR was detectable within luteal cells of CL. The MC2R mRNA levels were five-fold less abundant in day 9 than in day 4 CL (P<0.01). At both stages, ACTH agonist (ACTH 1-24) increased progesterone and prostaglandin (PG) E2 (PGE2) (P<0.01), but reduced PGF2α releases (P<0.01) in vitro. ACTH 1-24 injection increased plasma cortisol levels within 4h (P<0.01), but decreased (P<0.01) progesterone 24h later and for the following two days. ACTH administration to estrous rabbits caused a transitory increase in blood progesterone concentrations (P<0.01). Daily injections of ACTH did not modify progesterone profile following ovulation. In conclusion, ACTH directly up-regulates CL progesterone production in vitro via MC2R, but indirectly hampers luteal function via cortisol-GR associated mechanism.

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.

Progesterone receptor immunoreactivity differs in the uterus of pseudopregnant and medroxyprogesterone acetate-treated rabbits

Progesterone receptor (PR) plays an important role in mammals pregnancy which is characterized by greater progesterone plasma concentrations. We assessed PR protein distribution in the rabbit uterus by immunohistochemistry in two progestational conditions: pseudopregnancy (intact adult animals treated with hCG) and after application of a synthetic progestin, medroxyprogesterone acetate (MPA), to ovariectomized animals (OVX). PR immunoreactivity in uterine epithelium of pseudopregnant rabbits was increased in relation to non-pseudopregnant (NP) rabbits. Amounts were similar on Days 1, 3, and 5 of treatment, and was greater on Day 7 ( P < 0.001). In contrast, a significant diminution in PR immunoreactivity was observed in stroma cells from Days 1 to 7 ( P < 0.001). In OVX rabbits treated with MPA, an increase in PR immunoreactivity was observed in the uterine epithelium on Days 1 to 5 of treatment, reaching a maximum on Day 3 ( P < 0.001). In contrast, in stromal cells a diminution in PR immunoreactivity was observed when compared to the OVX group on Days 1, 3 and 7 of MPA treatment ( P < 0.001), and there was a slight increase on Day 5. Results suggest a differential time course and tissue specific immunoreactivity for PR in the uterus of the rabbit in two progestational conditions. The present study indicated synthetic progestins have different mechanisms of receptor regulation than those of natural hormones and it should be taken into account in reproductive applications.

Coating of Objects Introduced into the Oviduct of Pseudopregnant Rabbit Does

This investigation addresses the possibility of providing mouse embryos or other foreign objects with a protective mucin coat by transferring them into the oviduct of a live rabbit doe. Mouse embryos at the 8 or 16-cell stage, rabbit oocytes and latex spheres resembling mouse embryos in size were transferred to the ligated oviducts of ovulation-induced rabbit does. The does were killed 24 h later to have their oviducts flushed. A large proportion of the latex spheres (89%) and of the ovulated oocytes of the recipient does (92%) was recovered. The recovery rates for transferred rabbit oocytes, either intact or with the zona pellucida removed, were 61% and 51%, respectively, whereas that for mouse embryos was extremely poor (20%). Rabbit oocytes with or without zona were enveloped in a thick mucin coat regardless whether they had been transferred or ovulated by the recipients. The same applied to empty rabbit zonae. Mouse embryos and latex spheres were also covered by a mucin coat, but it was four times thinner. While residing in the rabbit oviduct, the mouse embryos continued developing to a stage comparable to what would have been expected in situ. During the subsequent in vitro culture, mouse embryos continued developing to the expanded blastocyst stage. They did, yet, not hatch from the zona. It may be concluded that particles of various origins, when placed into the oviduct of ovulated rabbit does, will be provided with a mucin covering which is, however, considerably thinner than that surrounding oocytes or zonae pellucidae originating from rabbits.

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.