Gap junctional connexin 37 is expressed in sheep ovaries

The objective of current study was to evaluate the expression of Cx37 in ovarian follicles and in corpora lutea (CL) during the estrous cycle in sheep. Ovine Cx37 was cloned and characterized to design speciesspecific probe and primers. In Exp. 1, ovaries were collected on d 13, 14, 15, and 16 of the estrous cycle, or from FSH-induced ewes at 0, 2, 4, 8, 12, 24, and 48 h after hCG treatment on d 15 of the estrous cycle. In Exps. 2 and 3, CL were collected on d 5, 10, and 15 of the estrous cycle, or at 0, 4, 8, 12, and 24 h after prostaglandin F_2α (PGF_2α)-induced luteal regression on d 10 of the estrous cycle, respectively. Ovarian tissues (e.g., granulosa cells, theca cells, ovarian follicles, and/or CL) were used for Cx37 immunostaining followed by image analysis or for determination of Cx37 mRNA expression by real-time RT-PCR. We demonstrated that (1) Cx37 protein was expressed in granulosa and cumulus oocyte complex compartments, ovarian blood vessels, and on the luteal cell borders, (2) expression of Cx37 mRNA was greater in granulosa than in theca cells of prevulatory follicles, (3) Cx37 mRNA expression in granulosa but not theca cells was affected by hCG treatment, (4) Cx37 protein and mRNA expression were dependent on the stage of luteal development, and (5) Cx37 expression changed during PGF_2α-induced luteal regression. Thus, Cx37 may play a role in follicular development and ovulation as well as in luteal tissue growth, differentiation, and regression.     

Cellular basis of luteal steroidogenesis in the human ovary

A primary monolayer cell culture system was developed to investigate human corpus luteum (CL) function in vitro. Steroidogenic cells were isolated by collagenase dispersal and Percoll density-gradient fractionation from CLs enucleated at progressive stages of the luteal phase (tubal surgery patients). 'Pure' granulosa-lutein cells were aspirated from ovulatory follicles at mid-cycle (in-vitro fertilization patients). The steroidogenic capacity (progesterone/20 alpha-dihydroprogesterone biosynthesis and aromatase activity) of isolated luteal cells was assessed in relation to CL development. Basal luteal cell steroidogenesis was maximal at around the expected time of ovulation and declined with CL age during the luteal phase. Conversely, human chorionic gonadotrophin (hCG)-responsive steroidogenesis was initially undetectable but developed as the luteal phase progressed. These results show that luteal cell steroidogenesis becomes increasingly dependent upon gonadotrophic support with CL age. This is evidence that functional luteolysis in human ovaries (1) is pre-programmed to occur at the cellular level, (2) is initiated automatically at the time of ovulation and (3) is reversed at the time of CL 'rescue' in early pregnancy by the direct action of trophoblastic hCG on steroidogenic luteal cells. The culture system described should be of value in further defining the control of human CL form and function at the cellular level.     

The expression of angiotensin and endothelin system members in bovine corpus luteum during estrous cycle and pregnancy

The aim of this study was to determine the changing profiles of the mRNA expression of members of angiotensin and endothelin system in bovine corpus luteum (CL) from different stages of the estrous cycle and pregnancy. Corpora lutea were accordingly assigned to the following stages; d 1–2, 3–4, 5–7, 8–12, 13–18, >18 (after regression) of estrous cycle and of early and late pregnancy (<4 and >4 mo). The block RT-PCR analysis of CL showed a significantly higher angiotensin converting enzyme (ACE) mRNA expression during mid and late luteal phases as well as after regression, but lower levels during pregnancy. Full quantitative real-time RT-PCR (LightCycler) confirmed this pattern of ACE mRNA expression. The angiotensin receptor type 1 (AT1R) mRNA expression was relatively stable throughout the periods examined. In contrast, AT2R mRNA temporarily decreased on d 8–12, followed by an increase to the highest levels during late luteal phase, and it remained at high levels during regression and pregnancy. Concentration of angiotensin II (Ang II) peptide in luteal tissue was highest after ovulation (d 1–2), decreased afterward, increased again during late luteal phase, and decreased to lower levels during regression and pregnancy. The mRNA expression and peptide concentration of endothelin 1 (ET-1) was high after ovulation followed by a decrease during mid and late luteal phases and increased again to the highest level after regression. The endothelin receptor type B (ETR-B) mRNA expression increased during late luteal phase and further after regression. In contrast, ETR-A and endothelin converting enzyme 1 (ECE-1) mRNA expression were relatively constant during all stages examined. In conclusion, the regulatory changes of both angiotensin and endothelin family members during early luteal phase and again during late luteal phase suggest a possible modulatory role of these vasoactive peptide families for bovine CL formation and regression.     

Ovarian features of the wild collared peccary (Tayassu tajacu) from the northeastern Peruvian Amazon

In this study, the ovaries of 27 wild collared peccaries (Tayassu tajacu) from the Amazonian region of northeastern Peru were examined macroscopically and microscopically, and expression of major steroidogenic enzymes was detected by immunohistochemistry. Our observations suggest a mean ovulation rate of 2.3 +/- 0.6 follicles and a low rate of reproductive wastage (0.4 +/- 0.6 oocytes or embryos per pregnancy). The collared peccary seems to exhibit follicular waves involving the synchronous growth of a cohort of follicles, several of which seem to attain selection. The presence of antral follicles in pregnant females suggests that follicular turnover continues during pregnancy. In cyclic animals, corpora lutea were characterised by the presence of distinct large and small luteal cell populations. The luteal volume in pregnant females was larger than that recorded for non-pregnant females. Through immunohistochemistry, it was observed that luteal cells from active corpora lutea exhibit intensive 3beta-HSD expression in advanced stages of pregnancy. This suggests that the corpora lutea seems to remain steroidogenically active throughout pregnancy and likely contribute to progesterone production during pregnancy.     

Expression of messenger ribonucleic acids that encode for 3 beta-hydroxysteroid dehydrogenase and cholesterol side-chain cleavage enzyme throughout the luteal phase of the macaque menstrual cycle

To study further the control of the primate corpus luteum, we obtained corpora lutea from cynomolgus macaques at defined stages of the luteal phase and examined steady state mRNA levels in these corpora lutea by Northern analysis for the two major enzymes involved in progesterone biosynthesis, cytochrome P450 cholesterol side-chain cleavage (P450SCC) and 3 beta-hydroxysteroid dehydrogenase (3 beta HSD). mRNAs for both P450SCC and 3 beta HSD were maximal or near maximal shortly after ovulation and luteinization (days 3-5 of the luteal phase). mRNA for P450SCC exhibited a slight, but nonsignificant (P greater than 0.05) decline throughout the remainder of the luteal phase and was undetectable upon luteal regression. Steady state levels of 3 beta HSD mRNA were significantly lower (P less than 0.05) from corpora lutea removed during the midluteal phase (days 7-8 of the luteal phase) than those in newly formed corpora lutea and declined to 10% of early luteal phase values by days 13-15 of the luteal phase. 3 beta HSD mRNA levels fell to nondetectable values upon luteal regression. These results reveal a paradoxical relationship between the steroidogenic activity of the primate corpus luteum in vivo and the steady state levels of the mRNAs that encode for the major enzymes involved in progesterone biosynthesis. Unlike serum progesterone concentrations, which are very low immediately after ovulation and then rise during the midluteal phase, the steady stale levels of P450SCC mRNA and 3 beta HSD appeared to be maximal or near maximal shortly after ovulation and declined throughout the remainder of the luteal phase. These findings are consistent with the notion that luteal lifespan is set at the time of ovulation and luteinization, and the decline in luteal function may be due in part to decay of specialized luteal cell mRNAs with finite half-lives.     

Characterization and autoradiographical localization of oxytocin receptors within the ovine endometrium in relation to post-partum corpus luteum function.

The induction of ovulation in early post-partum ewes is associated with a high incidence of premature luteal regression which is independent of the suckling stimulus but dependent on the stage post partum. The aim of the present study was to determine whether oxytocin receptors are present on uterine endometrium early in the luteal phase and hence ascertain whether oxytocin-induced uterine prostaglandin F2 alpha release is a possible mechanism involved in the premature regression of these post-partum corpora lutea. Ovarian and uterine tissues were collected on day 4 of the the cycle in ewes induced to ovulate at either 21 or 35 days post partum (n = 4 per group). A further four cyclic ewes were similarly synchronized to ovulate and acted as controls. Corpora lutea from the 21-day post-partum group were significantly (P less than 0.01) smaller, had a lower progesterone content and a reduced capacity to secrete progesterone in vitro than corpora lutea from 35-day post-partum or control ewes. A highly specific oxytocin receptor ligand 125I-labelled d(CH2)5[Tyr(Me)2, Thr4,Tyr-NH29]-vasotocin was used to localize and characterize high affinity oxytocin receptors in uterine endometrium (dissociation constant 145 pmol/l). Oxytocin receptor concentrations in endometrium from ewes induced to ovulate at 21 days post partum were on average five-fold higher (P less than 0.05) than in 35-day post-partum and control groups.     

Acid sphingomyelinase involvement in tumor necrosis factor alpha-regulated vascular and steroid disruption during luteolysis in vivo

TNF is well known for its role in inflammation, including direct effects on the vasculature. TNF also is implicated in the regulation of reproduction by its actions to affect ovarian steroidogenic cells and to induce apoptosis of corpus luteum (CL)-derived endothelial cells in vitro. We hypothesized that the disruption of TNF signaling would postpone the regression of the highly vascularized CL in vivo, and this effect could be replicated in mutant mouse models lacking TNF receptor (TNFRI(-/-)) and/or a critical enzyme of TNF signaling, acid sphingomyelinase (ASMase(-/-)). In the current study, the treatment of pseudopregnant mice with the luteolytic mediator prostaglandin F2-alpha (PGF) significantly increased TNF in the ovaries when compared with saline-treated controls. Treatment with PGF also reduced serum progesterone (P4) concentrations and caused involution of the CL. However, pretreatment of pseudopregnant mice with Etanercept (ETA), a TNF-neutralizing antibody, inhibited the PGF-induced decrease in P4 and delayed luteal regression. A similar outcome was evident in pseudopregnant TNFRI(-/-) animals. Treatment of luteal microvascular endothelial cells (MVECs) with TNF provoked a significant increase in ASMase activity when compared with the corresponding controls. Furthermore, TNF-induced MVEC death was inhibited in the ASMase(-/-) mice. The ASMase(-/-) mice displayed no obvious evidence of luteal regression 24 h after treatment with PGF and were resistant to the PGF-induced decrease in P4. Together these data provide evidence that TNF plays an active role in luteolysis. Further studies are required to determine the deleterious effects of anti-inflammatory agents on basic ovarian processes.     

Inhibition of delta-like ligand 4 induces luteal hypervascularization followed by functional and structural luteolysis in the primate ovary

Using specific inhibitors established that angiogenesis in the ovarian follicle and corpus luteum is driven by vascular endothelial growth factor. Recently, it has been demonstrated that the Notch ligand, delta-like ligand 4 (Dll4) negatively regulates vascular endothelial growth factor-mediated vessel sprouting and branching. To investigate the role of Dll4 in regulation of the ovarian vasculature, we administered a neutralizing antibody to Dll4 to marmosets at the periovulatory period. The vasculature was examined on luteal d 3 or d 10: angiogenesis was determined by incorporation of bromodeoxyuridine, staining for CD31 and cell death by staining for activated caspase-3. Ovulatory progesterone rises were monitored to determine effects of treatment on luteal function and time to recover normal cycles in a separate group of animals. Additionally, animals were treated in the follicular or midluteal phase to determine effects of Dll4 inhibition on follicular development and luteal function. Controls were treated with human IgG (Fc). Corpora lutea from marmosets treated during the periovulatory period exhibited increased angiogenesis and increased vascular density on luteal d 3, but plasma progesterone was significantly suppressed. By luteal d 10, corpora lutea in treated ovaries were significantly reduced in size, with involution of luteal cells, increased cell death, and suppressed plasma progesterone concentrations. In contrast, initiation of anti-Dll4 treatment during the midluteal phase produced only a slight suppression of progesterone for the remainder of the cycle. Moreover, Dll4 inhibition had no appreciable effect on follicular development. These results show that Dll4 has a specific and critical role in the development of the normal luteal vasculature.     

Luteal adenylyl cyclase does not develop sensitivity to desensitization by human chorionic gonadotropin in the absence of nonluteal ovarian tissue

There is evidence suggesting that the mere presence of a hormone-responsive adenylyl cyclase system in a tissue may not be sufficient for desensitization to occur since phosphorylation reactions might also be involved. The purpose of this study was to determine if luteal tissue in the absence of other ovarian tissues would desensitize to human CG (hCG). One or both ovaries were removed from rabbits 5 h before hCG-induced ovulation and the periovulatory follicles were transplanted underneath the kidney capsule where they formed ectopic corpus luteum [or corpora lutea (CL)]. Rabbits which were bilaterally ovariectomized received estradiol implants at the time of ovariectomy to maintain control serum estradiol concentrations. On day 7 of pseudopregnancy, the rabbits were injected with saline (control) or with 75 IU hCG and were killed 24 h later at which time ovarian and ectopic CL progesterone content and adenylyl cyclase activity were assessed. As expected, in ovarian CL there was decreased LH-responsive adenylyl cyclase (69% relative to control) and a correspondingly decreased luteal progesterone content (40% relative to control). In the same rabbits, the ectopic CL showed much the same pattern of response as the ovarian CL but perhaps to a slightly lesser extent (decreases relative to control of 59% in adenylyl cyclase response to LH and 29% in progesterone content). However, in rabbits with ectopic CL only, the luteal tissue showed no change either in hormone-responsive adenylyl cyclase activity or in progesterone content. Similarly, binding of radiolabeled hCG to luteal membranes 24 h after hCG was almost totally absent in ovarian CL, was decreased by 50% in ectopic CL with one ovary present, and was unaltered in ectopic CL of bilaterally ovariectomized rabbits. These data suggest that nonluteal ovarian tissue may be required for the induction in CL of the appropriate protein kinases for the proposed phosphorylations involved in adenylyl cyclase desensitization.     

Administration of a gonadotropin-releasing hormone agonist affects corpus luteum vascular stability and development and induces luteal apoptosis in a rat model of ovarian hyperstimulation syndrome

Ovarian hyperstimulation syndrome (OHSS) is a complication of ovarian stimulation with gonadotropins followed by the administration of human chorionic gonadotropin (hCG) to trigger the final steps of oocyte maturation. Gonadotropin-releasing hormone (GnRH) analogs are thought to be effective in preventing this complication and a clinical trial has found a lower incidence of OHSS in patients treated with these molecules. Our aim was to analyze the in vivo effect of a GnRH-I agonist on corpus luteum development and regression, ANGPT-1, ANGPT-2 and Tie-2 protein expression and luteal blood vessel stabilization, the expression of the steroidogenic acute regulatory protein (StAR) and the cytochrome P450 side-chain cleavage enzyme (P450scc) and cell proliferation, in ovaries from an OHSS rat model. To this end immature female Sprague-Dawley rats were hyperstimulated and treated with a GnRH-I agonist from the start of pregnant mare serum gonadotropin (PMSG) administration until the day of hCG injection for 5 consecutive days. Blood and tissue samples were collected 48h after hCG injection. Vascular endothelial growth factor VEGF levels were evaluated in the peritoneal fluid by ELISA. Serum progesterone and estradiol were measured by RIA. Histological features of sectioned ovaries were assessed in hematoxylin and eosin (H&E) stained slides. Luteal blood vessel stability, cell proliferation and apoptosis were assessed by immunohistochemistry for SMCA, PCNA, and TUNEL, respectively. P450scc, StAR, FLK-1, ANGPT-1, ANGPT-2, Tie-2 and PCNA protein levels were evaluated by Western blot from dissected corpora lutea (CL). The treatment with the GnRH-I agonist significantly decreased serum progesterone and estradiol levels as well as P450scc and StAR protein expression in the untreated OHSS group. In addition, the agonist significantly decreased the number of CL in the OHSS group, as compared with the untreated OHSS group. In the OHSS group, the area of periendothelial cells in the CL was larger than that of the control group. However, the treatment with the GnRH-I agonist significantly reduced the area of periendothelial cells in the CL in the OHSS group. The luteal levels of ANGPT-1 and its receptor Tie-2 significantly increased in the OHSS group when compared with the control group. Conversely, the administration of the GnRH-I agonist significantly decreased the levels of these factors in the CL from the OHSS group, as compared with the untreated OHSS group. In addition, the treatment with the GnRH-I agonist reduced the diameter of CL and decreased CL cell proliferation as compared with that observed in the untreated OHSS group. Finally, the GnRH-I agonist increased apoptosis in the CL from the OHSS group. In conclusion, these results show that GnRH-I agonist exerts diverse actions on the CL from a rat OHSS model. The decrease in P450scc, StAR, ANGPT-1 and Tie-2 expression, blood vessel stability and luteal proliferation leads to CL regression in the ovaries from OHSS rats. Moreover, our results suggest that the downregulation of ANGPT-1 and its receptor is a possible mechanism whereby GnRH-I agonists could prevent early OHSS.     

Ovarian responses of pregnant mare serum gonadotropin- and human chorionic gondotropin-primed rats: desensitizing, luteolytic, and ovulatory effects of a single dose of human chorionic gonadotropin.

We conducted a study to determine the morphological appearance and functional responsiveness of ovarian tissues after administration of hCG to 28-day-old rats primed 65 h earlier with PMS gonadotropin (PMSG) and after administration of a second dose of hCG 5 days later, i.e. to 33-day-old rats containing heavily luteinized ovaries. Sixty-five hours after the administration of 50 IU PMSG sc to 25-day-old rats, ovaries already contained an abundance of luteinized follicles and an adenylyl cyclase (AC) system that was responsive to LH, epinephrine, and NaF. The administration of 50 IU hCG sc at this time initially resulted in a loss of LH-responsive ovarian AC. Within 4 days of the hCG injection, the ovaries of the now 32-day-old rats were heavily luteinized, and ovarian AC was highly responsive to LH, epinephrine, and NaF. The administration of a single sc dose of 200 IU hCG to 33-day-old PMSC- and hCG-primed rats with luteinized ovaries resulted in a rapid desensitization of the ovarian AC to LH and a drop in serum progesterone levels, During the subsequent 7 days, serum progesterone levels continued to decline, while total ovarian AC reacquired responsiveness to LH by days 4--5 after the densensitizing dose of hCG. Dissection of ovarian components revealed, however, that the AC system of the corpora lutea originally present at the time of the second hCG injection remained permanently refractory to LH and that the AC in corpora lutea newly formed from freshly ovulated follicles exhibited a significant responsiveness to LH, epinephrine, and NaF. However, these new corpora lutea were not fully active, since serum progesterone never rose. Subcutaneous administration of 50 IU hCG to 33-day-old PMSG- and hCG-primed rats also promoted a rapid loss of AC responsiveness to LH. This lower concentration of hCG was not sufficient to promote follicular development or ovulation, and the ovarian AC remained refractory to LH for at least 7 days. Intravenous administration of 75 IU hCG to 33-day-old PMSG- and hCG-primed rats similarly promoted a rapid and permanent loss of luteal AC responsiveness to LH; again, follicles did not mature to a preovulatory state and, in fact, appeared to undergo atresia rather than ovulation. These results indicate that in heavily luteinized ovaries 1) hCG promotes desensitization of rat luteal AC to LH, 2) Desensitization of AC to LH stimulation in corpora lutea is permanent and irreversible, and 3) only under conditions where follicles mature and ovulate and new corpora lutea are formed does total ovarian AC reacqure responsiveness during the subsequent week.     

Expression and regulation of plasminogen activators,plasminogen activator inhibitor type-1, and steroidogenic acute regulatory protein in the rhesus monkey corpus luteum

The corpus luteum (CL) is a transient endocrine organ that secretes progesterone to support early pregnancy. Using primate materials obtained from rhesus monkeys, we have in this study investigated the expression and regulation of the plasminogen activators (PAs) and PA inhibitor type 1 (PAI-1) during CL development and regression. Adult (5-7 yr old) female rhesus monkeys were treated with pregnant mare serum gonadotropin/human chorionic gonadotropin to induce ovulation and follicular luteinization. At various luteal developmental stages, CL or whole ovaries were obtained for preparing luteal cells, Northern blot, in situ hybridization, and immunohistochemistry. We demonstrated that luteal cells from the rhesus monkey were able to produce both tissue type PA (tPA) and urokinase type PA, as well as the physiological PAI-1. During luteal development in the monkey, urokinase type PA was the major PA species taking part in the active angiogenesis and tissue remodeling processes in the forming CL. However, the mRNA as well as the enzymatic activity levels of tPA increased dramatically in monkey CL with the advent of luteolysis. This change of tPA levels was in a temporal coordination with the regulation of PAI-1 expression, resulting in an increased tPA activity at the initiation of luteolysis. Therefore, we suggest that tPA might be a luteolytic factor to the monkey CL. A PAI-1 modulated tPA activity might be important for the initiation of luteolysis in the monkey. In addition, we have also demonstrated that the expression of steroidogenic acute regulatory protein in the monkey CL was in accordance with the changes of progesterone production, suggesting that steroidogenic acute regulatory protein expression may be considered as a reliable marker for CL function in primates.     

Effects of the progesterone antagonist RU486 on ovarian activity in the rat

Adult female rats were treated for 2 or 4 weeks with the progesterone antagonist RU486 to study its effect on the regulation of ovarian function. In rats with 5-day ovarian cycles, the vaginal cyclicity disappeared. Uninterrupted vaginal cornification emerged within 4 days after the start of treatment and cornification persisted for the whole period of treatment. It took more than 2 weeks after cessation of 2-4 weeks of treatment before 5-day vaginal cycles reappeared. Ovarian weights increased rapidly resulting from the accumulation of large numbers of corpora lutea. In addition, the ovaries developed occasional follicular cysts which could reach an extremely large size (2 mm or more). Analysis of serial histological sections of ovaries, combined with plasma concentrations of estradiol-17 beta and progesterone, indicated cyclic ovulation and corpus luteum formation together with persistence of functional activity of already existing and newly formed corpora lutea. RU486 seems to have the unique property of dissociating cessation of luteal activity and ovulation in rats. After treatment with RU486, pituitary enlargement and mammary gland alveolar development were observed. It is hypothesized that these effects result from unopposed estrogen action on PRL secretion. The effects of RU486 are reversible: 4 to 5 weeks after the end of treatment ovarian activity seems normal (as evidenced by reduction of ovarian weights and 5-day vaginal cycles) except for the presence of occasional large follicular cysts which may require longer periods for their regression.     

Dynamic expression of caspase-2, -3, -8, and -9 proteins and enzyme activity, but not messenger ribonucleic acid, in the monkey corpus luteum during the menstrual cycle

Studies were designed to determine whether: 1) changes in caspase expression or activity occur in the macaque corpus luteum (CL) during its lifespan in the menstrual cycle, and 2) LH acting directly or via ovarian steroids regulates luteal caspases. Caspase-2, -3, -8, and -9 mRNAs were detectable by semiquantitative RT- or real time-PCR in CL, but levels did not differ between the early, mid, mid-late, late, and very-late luteal phases. Immunostaining for caspase-2 and -3 proteins was observed in luteal cells and appeared to peak by mid to mid-late stage. Enzyme activity for caspase-2, -3, -8, and -9 increased (P < 0.05) by mid-late stage, and then declined by the very-late stage. Treatment with GnRH antagonist + LH at the mid-late stage increased caspase-2, -8, and -9, but not -3, activity, compared with controls. Coadministration of a steroid synthesis inhibitor (trilostane) with GnRH antagonist + LH reduced (P < 0.05) caspase-2, -8, and -9 activity. Progestin (R5020) replacement during trilostane treatment did not restore caspase activity. Thus, initiator and effector caspases are present during CL development and regression in the menstrual cycle. The increased caspase activity at mid-late stage suggests that apoptosis is involved in early luteolysis in primates. Gonadotropin, perhaps via local steroids, modulates initiator caspases in the primate CL.     

Rabbit ovarian protein kinases. I. Effect of an ovulatory dose of human chorionic gonadotropin or luteinizing hormone on the subcellular distribution of follicular and luteal protein kinases.

Studies on the subcellular distribution of protein kinase activity in popped estrous follicles from rabbit ovaries revealed that 15% of the total cellular protein kinase activity was compartmentalized in the nuclear, mitochondrial, and microsomal fractions. About 50% of the particulate protein kinase activity was unaffected by the heat-stable protein kinase inhibitor and was thus cAMP-independent. The majority of cellular protein kinase activity was identified in the 105,000 X g supernatant fraction as cAMP-dependent. hCG- or coital-induced ovulation and subsequent corpus luteum (CL) formation, and hCG-induced luteal regression promoted changes and a redistribution of protein kinase activity among the subcellular fractions. In follicles, hCG promoted a transient decline of nuclear protein kinase activity as well as transient increases of the relative amount of protein kinases localized in the microsomal fractions before ovulation. In CL induced by a fertile mating, the specific activity as well as the total amount of protein kinases in the nuclear fraction were reduced 2-fold. Mitochondrial protein kinase activity from CL of pseudopregnancy and pregnancy was reduced 2-fold. The relative amount of protein kinase activity in microsomes of CL was increased 2-fold, but the specific activity was not affected. hCG-induced luteal regression resulted in a transient decline of the nuclear protein kinase activity in CL of 4-day pseudopregnant rabbits. In interstitial tissue, the specific activity of the nuclear protein kinase was increased over luteal levels, the mitochondrial-specific protein kinase remained at the reduced luteal levels, and the microsomal and cytosol protein kinase specific activities increased 2-fold. Studies with the heat-stable protein kinase inhibitor revealed that the hCG- or coital-induced redistribution of intracellular protein kinase affected both the cAMP-dependent and cAMP-independent activity to a similar degree and no changes of the relative distribution of cAMP-dependent vs. cAMP-independent activity were observed. These results indicate that the intracellular distribution and enzymatic activity of cAMP-dependent protein kinases in ovarian structures are subject to regulation by LH (hCG) and depend upon the various reproductive stages of the rabbit.     

Determination of secretion rates of estradiol, progesterone, oxytocin, and angiotensin II from tertiary follicles and freshly formed corpora lutea in freely moving sows

Two days before ovulation ovarian follicles of sows were implanted with microdialysis systems (MDS) which function like artificial capillaries with exteriorized inlets and outlets. Steroid hormones and paracrine acting factors such as oxytocin (OXT) and angiotensin II (AII) diffuse from ovarian tissue into the fluid, which is pumped through the MDS and collected in fractions. This allows determination of dynamic changes of estradiol (E2), progesterone (P), OXT, and AII secretion during the pre-, peri-, and postovulatory periods in freely moving sows. More than 80% of such implanted follicles ovulate and form competent corpora lutea (CL) allowing continuation of experimentation during the early luteal phase. Follicular E2 release increases before ovulation and decreases with increasing blood LH concentrations. Twenty to 30 h after beginning of the preovulatory LH surge P secretion increases gradually. Both peptides OXT and AII are released episodically by the preovulatory follicle. During the time of decreased E2 and not yet increased P secretion, i.e. during the periovulatory period, mean AII secretion was highest in comparison to the late follicular and early luteal phase. E2 remains measurable during the early luteal phase. OXT and AII were also topically applied into the follicular wall and after ovulation into the CL. AII had no effect on steroidogenesis of both structures. Although OXT was ineffective in the follicle, in young CL it stimulated P secretion. These results indicate that the MDS can be used to study late follicular and early luteal steroid and peptide secretion. The function of OXT and AII in the follicle remains obscure, whereas OXT has a luteotropic effect in young porcine CL.     

Bilateral maintenance of rabbit corpora lutea by the feto-placental unit

The present study was undertaken to determine whether the rabbit feto-placental unit maintains corpora lutea systematically and/or locally and the interrelationships between conceptus number, luteal weight, luteal progesterone concentrations and serum progesterone levels. Thirty-three does were divided into the following treatment groups: (I) bilaterally pregnant, two ovaries; (II) unilaterally pregnant, two ovaries; (III) bilaterally pregnant, one ovary; (IV) unilaterally pregnant, one ovary, contralateral and (V) unilaterally pregnant, one ovary, ipsilateral. Blood samples were obtained from all rabbits on days 6, 9, 12, 15, 18 and 21 post coitum. Does were killed on day 21, and the percentage of viable fetuses, fetal weights, and luteal weights recorded. Blood samples and corpora lutea were analysed for progesterone. Serum progesterone levels were similar for all groups until day 9 post coitum. Levels in groups III, IV and V declined significantly between days 9 and 12 following removal of one ovary at day 9. Fetal viability, fetal weights and luteal progesterone concentrations did not differ among any of the groups. Luteal weights did not differ among groups I, III, IV and V, but luteal weights of animals in group II were lower than those of group I (P less than 0.05). Ratios of viable fetuses to number of corpora lutea ranged from 1:11-10:5. No differences were observed in serum progesterone, luteal weights or luteal progesterone concentrations among animals with two conceptuses and those with seven or more, but serum progesterone levels in does with only one conceptus were lower than those in does with more (P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)