Midluteal Phase Corpora Lutea Research Articles

Effect of Tumor Necrosis Factor-α on in vitro Prostaglandin Production in Buffalo Corpus Luteum

Background: Corpus luteum plays key role in embryonic survival. Prostaglandins are the important regulator controlling the life span of corpus luteum. The present study investigated the effect of various doses of TNFα on in vitro PGF2α and PGE2 production and expression profiling of PGFS and PGES mRNA in buffalo Corpus Luteum (CL).Methods: Buffalo ovaries with mid-luteal phase CL were collected from the abattoir and CL were enucleated from surrounding tissues. Corpus luteum were finely chopped, rinsed with HBSS (Hanks Balanced Salt Solution) medium; supplemented with gentamycin and 0.1% BSA and incubated at 37°C for 1 hr in HBSS containing 0.1% collagenase. The cell suspension following filtration was washed by HBBS supplemented with gentamycin and 0.1% BSA (bovine serum albumin) and was treated with increasing doses of TNFα (0.1, 0.5 and 1.0 nM) and cultured at 38.5°C, 5% CO2 level for 24 hr. Result: There was dose dependent increase in concentrations of PGF2α and PGE2 with increasing doses of TNFα. The PGFS (prostaglandin F synthase) mRNA expression increased with increasing doses of TNFα. However, there was decrease in PGES (prostaglandin E synthase) mRNA expression at 0.1 nM and 0.5 nM TNFα but PGES mRNA expression increased at 1.0 nM TNFα as compared to control. It can be concluded that TNFα may alter PGES and PGFS mRNA expression and prostaglandin secretion in buffalo CL.

Modulatory role of leptin on ovarian functions in water buffalo (Bubalus bubalis)

The aim of the present study was to demonstrate the modulatory role of leptin on bubaline granulosa cells (GCs) and luteal cells (LCs) functions using an in vitro cell culture system and to establish a cross talk between leptin and insulin-like growth factor-1 (IGF-1). GCs were collected from group IV follicles (>13 mm size) and LCs from mid-luteal phase corpus luteum and were grown in serum-containing media supplemented with leptin at three different dose rates (0.1, 1, and 10 ng/mL) and time durations (24, 48, and 72 hours). We evaluated the production and secretion of estradiol (E2) and progesterone (P4) using RIA and the mRNA expression of steroidogenic acute regulatory protein (STARD1), cytochrome P450 cholesterol side-chain cleavage (CYP11A1), 3β-hydroxysteroid dehydrogenase (3β-HSD), cytochrome P450 aromatase (CYP19A1), sterol regulatory element–binding protein 1 (SREBP1), steroidogenic factor-1 (SF1), anti-apoptotic gene PCNA, pro-apoptotic gene caspase 3 and endothelial cell marker, Von Willebrand factor (vWF), using quantitative real-time polymerase chain reaction. The results depicted a direct inhibitory action of leptin on GCs steroidogenesis in a time-dependent manner (P < 0.05), whereas in the presence of IGF-1 the inhibitory effect was reverted. Furthermore, leptin augmented both cellular proliferation (PCNA) and apoptosis (caspase 3). On the other hand, in LCs, leptin alone showed an apparent stimulatory effect on steroidogenesis (P < 0.05); however, in the presence of IGF-1, an antagonistic effect was witnessed. Moreover, leptin had an inhibitory effect on apoptosis while promoted cellular proliferation and angiogenesis. These findings were further strengthened by immunocytochemistry. To conclude, these observations for the first time reported that in buffaloes leptin has a direct dose-, time-, and tissue-dependent effect on ovarian steroidogenesis, angiogenesis, and cytoprotection, and furthermore, it can regulate the effect of systemic factors like IGF-1. Hence, this in vitro study provides an insight into the putative roles of leptin alone and its interactions in vivo.

Decreased cholesterol uptake and increased liver x receptor-mediated cholesterol efflux pathways during prostaglandin F2 alpha-induced and spontaneous luteolysis in sheep.

In nonprimate species, it has been well established that prostaglandin F2 alpha (PGF2alpha) initiates luteolysis. Changes in intracellular cholesterol concentrations caused by modulation of cholesterol uptake and efflux may mediate PGF2alpha-induced luteolysis. These changes in cholesterol efflux and uptake are controlled, in part, by the liver x receptors (LXR) alpha (NR1H3) and beta (NR1H2), nuclear receptors that increase expression of genes necessary for cholesterol efflux or limiting cholesterol uptake. Therefore, we hypothesized that PGF2alpha reduces expression of cholesterol uptake and increases expression of cholesterol efflux genes, mediated in part by enhanced LXR activity. To test this hypothesis, an induced luteolysis model was used whereby ewes were treated during their midluteal phase with saline or PGF2alpha and corpora lutea (CL) collected 12, 24, or 48 h later for determination of mRNA and protein concentrations by quantitative real-time PCR and Western blot analysis, respectively. As a complementary approach, CL undergoing spontaneous luteolysis were compared to midluteal phase CL. The lipoprotein receptors responsible for cholesterol uptake were significantly decreased in both luteolysis models. Expression of the LXR target gene ATP binding cassette subfamily A1 (ABCA1), an important mediator of cholesterol efflux, was significantly increased in both experimental models. Chromatin immunoprecipitation confirmed that PGF2alpha treatment resulted in enhanced NR1H3 and NR1H2 binding to the ABCA1 promoter. Qualitative changes in lipid droplet distribution were also observed following PGF2alpha treatment. These data support the hypothesis that reduced cholesterol uptake and increased efflux mediate luteolysis in sheep, which is partially controlled by PGF2alpha stimulation of LXR activity.

STARD6 is expressed in steroidogenic cells of the ovary and can enhance de novo steroidogenesis

STARD6 is a member of the StAR-related lipid transfer (START) domain family of proteins whose function thus far remains obscure. While it recently was shown to facilitate steroidogenesis in a cell-free setting, it has not been localized to steroidogenic cells of normal reproductive tissues. In a recent microarray study, we detected STARD6 mRNA in cultured porcine ovarian granulosa cells which are steroidogenic. In the present study, we examined regulation of STARD6 mRNA in porcine granulosa cultures, and found that it was not regulated by cyclic AMP, but it was reduced by combined knockdown of the transcription factors GATA4 and GATA6. We detected both STARD6 mRNA and protein in fresh granulosa cells and whole antral follicles and different stage corpora lutea of pig. The highest levels were discovered in the mid-luteal phase corpus luteum. Immunolocalization within ovarian tissues indicated robust STARD6 immunoreactivity in steroidogenic cells of the corpus luteum. Relatively lesser amounts of STARD6 signal were found in granulosa cells, theca cells, and oocytes. To test the ability of STARD6 to facilitate de novo steroidogenesis, non-steroidogenic COS-1 cells were co-transfected with components of the P450 cholesterol side-chain cleavage system, enabling them to make pregnenolone, and STARD6. STARD6 increased pregnenolone production by two- to three-fold over the empty vector control. In summary, STARD6 is found in the pig ovary, exhibits the strongest expression in highly steroidogenic luteal cells, and significantly enhances pregnenolone production in transfected COS cells independent of cyclic AMP treatment. Collectively, these findings indicate that STARD6 may contribute to steroidogenesis in ovarian cells, but also suggests other cellular functions that require cholesterol trafficking.

Nitric oxide stimulates progesterone and prostaglandin E 2 secretion as well as angiogenic activity in the equine corpus luteum

Cytokines and nitric oxide (NO) are potential mediators of luteal development and maintenance, angiogenesis, and blood flow. The aim of this study was to evaluate (i) the localization and protein expression of endothelial and inducible nitric oxide synthases (eNOS and iNOS) in equine corpora lutea (CL) throughout the luteal phase and (ii) the effect of a nitric oxide donor (spermine NONOate, NONOate) on the production of progesterone (P4) and prostaglandin (PG) E 2 and factor(s) that stimulate endothelial cell proliferation using equine luteal explants. Luteal tissue was classified as corpora hemorrhagica (CH; n = 5), midluteal phase CL (mid-CL; n = 5) or late luteal phase CL (late CL; n = 5). Both eNOS and iNOS were localized in large luteal cells and endothelial cells throughout the luteal phase. The expression of eNOS was the lowest in mid-CL ( P < 0.05) and the highest in late CL ( P < 0.05). However, no change was found for iNOS expression. Luteal explants were cultured with no hormone added or with NONOate (10 −5 M), tumor necrosis factor-α (TNFα; 10 ng/mL; positive control), or equine LH (100 ng/mL; positive control). Conditioned media by luteal tissues were assayed for P4 and PGE 2 and for their ability to stimulate proliferation of bovine aortic endothelial cells (BAEC). All treatments stimulated release of P4 in CH, but not in mid-CL. TNFα and NONOate treatments also increased PGE 2 levels and BAEC proliferation in CH ( P < 0.05). However, in mid-CL, no changes were observed, regardless of the treatments used. These data suggest that NO and TNFα stimulate equine CH secretory functions and the production of angiogenic factor(s). Furthermore, in mares, NO may play a role in CL growth during early luteal development, when vascular development is more intense.

Is FAS/Fas Ligand System Involved in Equine Corpus Luteum Functional Regression?1

Proapoptotic factor Fas ligand (FASL) and its cell surface receptor FAS are tumor necrosis factor superfamily members that trigger apoptosis in different cell types. However, their influence on luteal steroidogenesis is not clearly understood. The aim of the present work was to determine (i) the presence of the cytokine FASL and its receptor FAS in the mare's corpus luteum (CL) throughout the luteal phase, as well as (ii) the influence of FASL alone, or together with the cytokines tumor necrosis factor alpha (TNF) and interferon gamma (IFNG), on equine luteal cell production of luteotrophic and luteolytic factors, cell viability, and apoptosis. FASL and FAS protein expression and mRNA transcription were evaluated in different luteal stages of the equine CL by Western blotting and real-time PCR assays, respectively. Protein expression and FASL mRNA transcription increased in the late CL. Also, FAS and FASL proteins were present in large steroidogenic and endothelial CL cells throughout the luteal phase, as demonstrated by immunohistochemistry. Equine luteal cells isolated from midluteal phase CL were stimulated without (control) or with exogenous cytokines: FASL (10 ng/ml); TNF+IFNG (10 ng/ml each; positive control) or FASL+TNF+IFNG (10 ng/ml each). FASL clearly inhibited in vitro progesterone and prostaglandin E(2) (PGE(2)) production by equine luteal cells but increased prostaglandin F(2alpha) (PGF(2alpha)). Furthermore, FASL effect on equine luteal cell viability depended on the presence of cytokines TNF and IFNG. In conclusion, this study shows the presence of FASL and FAS in the equine CL and suggests their importance in functional luteolysis.

The human corpus luteum: life cycle and function in natural cycles

To summarize recent advances in the understanding of the endocrine signaling pathways between the hypothalamus, pituitary, and human corpus luteum (CL); to examine the major paracrine and autocrine mechanisms and the key genes and proteins involved in CL development, function, and regression in natural cycles; to review the endocrine and molecular response of the midluteal phase CL to in vivo administration of human chorionic gonadotropin (hCG); and to describe the ultrasonographic and Doppler evaluation of the ovary and endometrium throughout the luteal phase. Published data in the literature, including the basic and clinical research studies of the authors. University-affiliated hospital and research centers. None. None. Clinical and molecular analysis of human CL function. The endocrine function of the subpopulations of luteal cells is critical for the maintenance of CL function, including neovacularization and steroid hormones production. We consider the key genes and proteins that favor development of luteal structure and function throughout the menstrual cycle and in our model of hCG treatment resembling early pregnancy. These data indicate that the functional lifespan of the CL depends on paracrine and autocrine mechanisms. Therefore, the significance of the key genes and proteins that we analyze in lutein cells during CL development, function, demise, and rescue by hCG is likely to bring new therapeutic applications for the management of fertility defects and the control of fertility.

Progesterone and Caspase‐3 Activation in Equine Cyclic Corpora Lutea

Soon after ovulation, the newly formed corpus luteum (CL) starts secreting progesterone (P(4)), necessary for implantation. The CL, an ovarian transient endocrine organ, undergoes growth and regression throughout its life span. The objective of this study was to evaluate if caspase-3 mediates cell death in the equine cyclic luteal structures and relate it to luteal endocrine function. Blood and luteal tissue were collected during the breeding season after slaughter from 38 randomly assigned cycling mares. Luteal tissues were classified as corpora haemorrhagica (CH; n = 7); mid luteal phase corpora lutea (Mid-CL; n = 17); late or regressing corpora lutea (Late-CL; n = 9) and corpora albicans (CA; n = 5). Plasma P(4) concentration, determined by radioimmunoassay, showed a significant increase from CH to Mid-CL (p < 0.001), followed by a decrease to Late-CL (p < 0.001) and CA (p < 0.001). Caspase-3 processing and poly (ADP) ribose polymerase (PARP) degradation were assessed by western blotting. Active caspase-3 was twofold increased in Mid-CL, Late-CL and CA as compared with CH (p < 0.05). Immunocytochemistry also showed a significant increase in caspase-3 expression in large luteal cells in all structures when compared with CH (p < 0.05). Consistently, the endogenous caspase-3 substrate, PARP, was markedly degraded from CH to CA (p < 0.05). In fact, the ratio of full-length to degraded PARP showed a significant decrease from CH to Mid-CL, Late-CL and CA (p < 0.05). Finally, the decrease in P(4) from Mid- to Late-CL coincided with no further increases in apoptosis. In conclusion, these results suggest that the effector caspase-3 of apoptosis, might play an important role during luteal tissue involution in the mare, even though its relationship with P(4) remains to be elucidated.

Identification of novel genes regulated by LH in the primate corpus luteum: insight into their regulation during the late luteal phase.

The process of luteinization, during which granulosa cells are transformed into luteal cells, is accompanied by dramatic changes in the response of luteal cells to LH. Although luteal cells require LH-cAMP signalling cascade for survival, whether these cells respond to trophic factors through changes in gene expression remains poorly characterized. In an attempt to characterize gonadotrophin (LH)-regulated gene expression in the bonnet monkey corpus luteum (CL), changes in gene expression after GnRH antagonist treatment to inhibit LH secretion, different stages of CL and during hCG-simulated early pregnancy were examined using differential display RT-PCR, Northern blot and semiquantitative RT-PCR analyses. We have identified seven non-redundant cDNA's whose expression were regulated by LH. The results show that inhibition of LH secretion not only leads to down-regulation in the expression of genes, e.g. low density lipoprotein (LDL) receptor and Aldose reductase, but expression of some of the genes was up-regulated, e.g. Humanin, RNA helicase, Lyric protein, Acidic ribosomal phosphoprotein and KIAA1750. mRNA levels of the genes identified as up-regulated after LH inhibition were higher during late compared to the early and mid-luteal phase CL, but treatment with hCG down-regulated their expressions. We conclude that we have identified novel genes (known and unknown) that are up or down-regulated by LH, and the results suggest that LH-mediated activation and repression of expression of many genes is central to the regulation of the structure and function of the CL in the monkey.

Expression and distribution of AP-1 transcription factors in the porcine ovary.

The activator protein-1 (AP-1) transcription factors are important regulators of cell proliferation and differentiation. The developmental distribution of AP-1 family members in porcine ovary has not been previously investigated. We examined the expression of AP-1 factors in porcine ovarian follicles, granulosa cells, and corpora lutea at different stages of development. Immunoblot analyses confirmed that c-Jun, JunD, JunB, c-Fos, Fra-1, Fra-2, and FosB immunoreactive proteins were present in whole-cell extracts (WCE) of all antral follicles and midluteal phase corpora lutea (CL) as well as granulosa cells (GC) isolated from different-sized antral follicles. The intensities of c-Jun and c-Fos protein bands were decreased in CL WCE compared to antral follicles. In granulosa cells from preovulatory follicles (8-10 mm), Fra-2 exhibited a shift from 43 kDa to 46 kDa when compared to granulosa cells from smaller antral follicles. Separation of cytoplasmic and nuclear extracts was performed to determine if developmental differences between these fractions existed. Most AP-1 factors predominated in the nuclear fraction with notable exceptions. c-Fos predominated in the nucleus in GC and follicles but predominated in the cytoplasmic fraction of CL. With the exception of GC from 1-2-mm follicles, in which expression was similar between fractions, Fos-B was found predominantly in the cytoplasmic fraction. Fra-1 exhibited similar expression between cytoplasmic and nuclear fractions for all tissues. Immunohistochemical (IHC) analyses of porcine ovary sections were performed to determine the cellular distribution of these factors at different follicular stages, and immunopositive nuclei were evaluated. In primordial and primary unilaminar follicles, all AP-1 factors studied except for FosB were detected in granulosa nuclei. Granulosa cell nuclei of multilaminar preantral follicles were immunopositive for all factors, with lower expression of FosB. Antral follicles exhibited GC and thecal cell nuclear staining for all factors with the exception of FosB in theca. Luteal cells exhibited the most intense nuclear staining for JunD and Fra-2, whereas all other factors were present in luteal cell nuclei although to a lesser extent. IHC with FosB antibodies yielded mostly cytoplasmic staining but only weak luteal nuclear staining. In corpora albicantia, low levels of staining were seen for all AP-1 factors. The DNA-binding abilities of these factors in granulosa cells and CL were evaluated by EMSA. Nuclear extracts from granulosa cells from 1-2-mm or 8-10-mm antral follicles bound an AP-1 DNA consensus sequence and complexes consisted predominantly of c-Jun, JunD, JunB, c-Fos, and Fra-2. In CL, c-Jun, JunD, JunB, and Fra-2 were present in DNA-binding complexes, and c-Fos binding was not detected. In conclusion, our results suggest that expression and DNA-binding activity of AP-1 factors in follicular structures changes with luteinization. Differentiation to the luteal phenotype involves a reduction in nuclear c-Jun and c-Fos and a predominance of JunD and Fra-2.

GATA-4 and GATA-6 transcription factors: expression, immunohistochemical localization, and possible function in the porcine ovary.

The expression and localization of GATA-4 and GATA-6 mRNAs and proteins were assessed in porcine ovaries at different stages of the estrous cycle. Reverse transcription polymerase chain reaction and Western blot analyses revealed that GATA-4 and GATA-6 transcripts and proteins were strongly expressed in granulosa cells isolated from antral follicles, intact antral follicles, corpora hemorrhagica (CH), and midluteal phase corpora lutea (CL). Immunoblot analyses showed two predominant proteins with molecular masses of approximately 53 and 55 kDa for GATA-4 and one 55-kDa protein for GATA-6. Immunohistochemical studies revealed GATA-4 and GATA-6 nuclear staining in granulosa cells of healthy primordial and primary antral follicles and antral follicle of various sizes. The percentage of immunopositive thecal cell nuclei increased with follicular development. In CH and CL, luteal cells displayed nuclear immunoreactivity for both transcription factors. Regressing CL showed a decrease in GATA-immunopositive cells. Immunoreactivity for GATA-4 and GATA-6 was present in most blood vessels. In electrophoretic mobility shift assays, nuclear protein extracts isolated from granulosa cells and CL exhibited both GATA-4 and GATA-6 binding to a GATA consensus oligonucleotide, with GATA-4 the predominant binding protein. GATA-4 and GATA-6 DNA binding was elevated in granulosa cell nuclear extracts from preovulatory (8-10 mm) follicles. Cotransfection of primary cultures of luteinizing granulosa cells with GATA-4 or GATA-6 expression vectors increased the activity of the porcine steroidogenic acute regulatory protein gene promoter significantly but did not significantly activate the inhibin alpha gene promoter. The detection of GATA-4 and GATA-6 mRNAs and proteins in porcine ovaries and the identification of at least one possible target gene may help to establish roles for these GATA factors in follicular development and luteal function.

Cloning and characterization of porcine ovarian estrogen receptor beta isoforms.

The cDNA for the full-length porcine estrogen receptor beta (ER beta) and an alternatively spliced transcript with a deletion of exon 5 (ER beta delta 5) was cloned from pig ovary. RNase protection assays revealed that ER beta mRNA was expressed in the preovulatory follicles and early, midluteal, and regressing corpora lutea (CL) of eCG +/- hCG-primed gilts. ER beta and ER beta delta 5 transcripts were shown by semiquantitative reverse transcription polymerase chain reaction to be expressed at a ratio of approximately 2:1 in granulosa cells, small, medium, and large antral follicles, and midluteal phase corpora lutea of unprimed animals. Immunoreactive ER beta proteins corresponding to the size of in vitro translated ER beta and ER beta delta 5 were detected by immunoblot. Full-length ER beta was detected in granulosa, small, medium, and large antral follicles, and midluteal phase CL of unprimed animals. Putative ER beta delta 5 immunoreactive bands were abundant only in granulosa cell extracts. In COS-1 cells, transfected ER beta delta 5 had no effect on basal transcription of an estrogen-responsive reporter construct but did repress wild-type ER beta transactivation when cotransfected at 10-fold excess plasmid. No repression of ER alpha transactivation was observed. In primary granulosa cell cultures, transfected ER beta delta 5 plasmid did not inhibit basal reporter activation. ER beta delta 5 was shown by immunofluorescence to localize to the nucleus in transfected COS-1 cells. In vitro translated ER beta delta 5 proteins bound estrogen response elements in DNA in electrophoretic mobility shift assays, as indicated by supershift analysis. ER beta is abundant in porcine ovary, and a naturally occurring splice variant missing exon 5 may have biological function.

Distinct cellular localization and regulation of endothelin-1 and endothelin-converting enzyme-1 expression in the bovine corpus luteum: implications for luteolysis.

Endothelin-1 (ET)-1 within the corpus luteum (CL) is rapidly up-regulated during natural or PGF(2 alpha)-induced luteolysis; however, such an increase was not observed at early luteal stage, when the CL is refractory to PGF(2 alpha). The mature and active form of ET-1 is derived from the inactive intermediate peptide, big ET-1, by ET-converting enzyme (ECE)-1. This study therefore examined the developmental and cell-specific expression of ECE-1 in bovine CL. A significant, 4-fold, elevation in ECE-1 expression (mRNA and protein levels) occurred during the transition of the CL from early to midluteal phase. Analysis using in-situ hybridization and enriched luteal cell subpopulations showed that both steroidogenic and endothelial cells of the CL expressed high levels of ECE-1 mRNA; prepro ET-1 mRNA, on the other hand, was only expressed by resident endothelial cells. These data suggest that luteal parenchymal and endothelial cells may cooperate in the biosynthesis of mature bioactive ET-1. In the mature CL, ECE-1 mRNA increase occurred both in steroidogenic and endothelial cells and was accompanied by a significant rise in ET-1 peptide. However, in contrast to ECE-1, prepro ET-1 mRNA levels were similar in early and midluteal-phase CL. Low ECE-1 levels during the early luteal phase, restricting the production of active ET-1, may explain why the immature CL is able to withstand PGF(2 alpha)-induced luteolysis.

Expression of steroidogenic acute regulatory protein in the human corpus luteum throughout the luteal phase.

The expression of the steroidogenic acute regulatory protein (StAR) in the human corpus luteum (CL) was examined throughout the luteal phase. The primary 1.6-kb StAR transcript was in greater abundance in early (3.1-fold) and mid (2.2-fold) luteal phase CL compared with late luteal phase CL. The larger StAR transcript (4.4 kb) was found in early and midluteal phase CL, but was not detected in late luteal phase specimens. Mature StAR protein (30 kDa) was present in lower amounts within late CL compared with early and midluteal phase CL. The StAR preprotein (37 kDa) was also detected in greater abundance in early and midluteal CL. Immunohistochemistry revealed that StAR staining was most prominent in thecal-lutein cells throughout the luteal phase. The intensity of the signal for StAR exhibited significant changes throughout the luteal phase, being most intense during the midluteal phase and least during the late luteal phase. Plasma progesterone concentrations were highly correlated (r = 0.73 and r = 0.79) with luteal expression of the preprotein and mature StAR isoforms, respectively, throughout the luteal phase. To examine the LH dependency of StAR expression, the GnRH antagonist, Cetrorelix, was administered during the midluteal phase. Cetrorelix caused a decline in serum LH levels within 2 h, which, in turn, caused a pronounced decline in plasma progesterone within 6 h. The StAR 4.4-kb transcript was not detectable, and the 1.6-kb transcript was reduced by approximately 50% within 24 h of Cetrorelix treatment. The mature 30-kDa StAR protein level declined approximately 30% after Cetrorelix treatment. We conclude that 1) StAR mRNA and protein are highly expressed in early and midluteal phase CL; 2) StAR protein is present in both thecal-lutein and granulosa-lutein cells throughout the luteal phase; 3) StAR protein levels in the CL are highly correlated with plasma progesterone levels; 4) declining StAR mRNA and protein levels are characteristic of late luteal phase CL; and 5) suppression of LH levels during the midluteal phase results in a marked decline in plasma progesterone and a diminished abundance of StAR transcripts in the CL without a corresponding significant decline in StAR protein. Collectively, these data are consistent with the idea that StAR gene expression is a key determinant of luteal progesterone during the normal menstrual cycle. However, the pharmacologically induced withdrawal in the midluteal phase of LH support diminishes luteal progesterone output by mechanisms others than reduced StAR protein levels.

Expression of Bcl-2 and Bax in the human corpus luteum during the menstrual cycle and in early pregnancy: regulation by human chorionic gonadotropin.

To investigate the relationship between apoptosis and the Bcl-2/ Bax system in the human corpus luteum (CL), the frequency of apoptosis and expression of Bcl-2 and Bax were examined in the CL during the menstrual cycle and in early pregnancy. In situ analysis of DNA fragmentation showed that the number of apoptotic cells was much greater in the regressing CL than that in the midluteal phase CL, whereas there were almost no apoptotic cells in the CL of early pregnancy. Immunohistochemistry revealed that Bcl-2 expression was observed in the luteal cells in the midluteal phase and early pregnancy, but not in the regressing CL. In contrast, Bax immunostaining was observed in the regressing CL, but not in the midluteal phase and early pregnancy. bcl-2 messenger ribonucleic acid (mRNA) levels in the CL during the menstrual cycle were highest in the midluteal phase and lowest in the regressing CL. In the CL of early pregnancy, bcl-2 mRNA levels were significantly higher than those in the midluteal phase. In contrast, bax mRNA levels were highest in the regressing CL and remarkably low in the CL of early pregnancy. Western blot analyses revealed that Bcl-2 expression was significantly lower in the regressing CL than in the midluteal phase and early pregnancy, and that Bax expression was, in contrast, significantly higher in the regressing CL than in the midluteal phase and was remarkably low in the CL of early pregnancy. When corpora lutea of the midluteal phase were incubated with hCG, hCG significantly increased the mRNA and protein levels of Bcl-2 and significantly decreased those of Bax. In conclusion, Bcl-2 and Bax may play important roles in the regulation of the life span of the human CL by controlling the rate of apoptosis. hCG may act to prolong the life span of the CL by increasing Bcl-2 expression and decreasing Bax expression when pregnancy occurs.

Expression of adrenomedullin in the human corpus luteum

Objective: Adrenomedullin (AM) is a potent hypotensive peptide found in human pheochromocytoma tissue. In the present study, the expression of AM mRNA in the human ovary was examined. Design: Ovarian mRNA was analyzed in the follicle, the corpus luteum of mid-luteal phase, and early pregnancy. Setting: Gunma University School of Medicine, Gunma, Japan. Patient(s): Premenopausal women with histologically normal ovary who were undergoing salpingoophorectomy. Intervention(s): The dominant follicle and corpora lutea were isolated and total RNA was extracted from these tissues. Main Outcome Measure(s): Northern blot analysis of AM, receptor activity–modifying protein 2 (RAMP2), and LH/hCG receptor mRNA in human samples. Result(s): An AM mRNA transcript of 1.6 kilobases (kb) was detected in corpus luteum tissue; this transcript was identical to that which has been detected in placenta and fetal membrane. The AM and LH/hCG receptor mRNA levels were low in the mature follicle but increased in the corpus luteum of the mid-luteal phase and were maintained during early pregnancy. A single transcript of 0.8 kb for RAMP2 was also seen in the follicle and corpus luteum, the level of RAMP2 mRNA was relatively high in the preovulatory follicle and RAMP2 was present in the corpus luteum. Conclusion(s): The expression of AM, its receptor, and LH/hCG receptor may be an important component in the process of development and differentiation of the corpus luteum.

Superoxide dismutase expression in the human corpus luteum during the menstrual cycle and in early pregnancy

To investigate the possible role of the superoxide radical and its scavenging system in the human corpus luteum, superoxide dismutase (SOD) values and lipid peroxide concentrations were analysed in the corpora lutea during the menstrual cycle and in early pregnancy. Copper-zinc SOD (Cu,Zn-SOD) activities increased from the early to mid-luteal phase, and gradually decreased thereafter and were the lowest in the regression phase. In pregnant corpus luteum, Cu,Zn-SOD activities were significantly higher than those in the mid-luteal phase. In contrast, manganese SOD (Mn-SOD) activities were low in the mid-luteal phase and increased toward the regression phase. Changes in mRNA expression of both types of SOD were similar to changes in their activities. Lipid peroxide concentrations were the highest in the regression phase whereas they were remarkably low in pregnant corpus luteum. The effects of human chorionic gonadotrophin (HCG) on luteal SOD were studied in vitro. HCG significantly increased Cu,Zn-SOD expression in mid-luteal phase corpora lutea, but not in late luteal phase corpora lutea. In conclusion, the present study suggests that the superoxide radical and its scavenging system, especially Cu,Zn-SOD, play important roles in the regulation of human luteal function. The stimulation of luteal Cu, Zn-SOD expression by HCG may be important in maintaining luteal cell integrity when pregnancy occurs.

Ubiquitin and apoptosis in the corpus luteum of the marmoset monkey (Callithrix jacchus).

The polypeptide ubiquitin covalently binds to cytoplasmic proteins and marks them for proteolytic degradation. Ubiquitin is upregulated during apoptosis in some systems. Apoptosis increases during luteolysis but it is not known whether ubiquitin is expressed in regressing corpora lutea. Marmoset ovaries were removed on day 10 of the luteal phase from animals that had received either no treatment, treatment with the PGF2 alpha analogue cloprostenol 24 h earlier, or treatment with the GnRH antagonist antarelix for either 24 or 48 h before ovary collection. Ubiquitin was localized on ovarian sections by immunocytochemistry, and oligonucleosome formation characteristic of apoptosis was examined in isolated corpora lutea by electrophoresis of extracted [32P]DNA. Oligonucleosome formation was low in midluteal corpora lutea on day 10 but increased after induced luteal regression with PGF2 alpha and GnRH antagonist. Nuclear ubiquitin immunoreactivity was found in 1.66 +/- 0.66 steroidogenic cells and cytoplasmic staining was found in 0.4 +/- 0.3 steroidogenic cells (per x 40 field of view) in midluteal phase corpora lutea on day 10. Luteolytic induction with PGF2 alpha significantly increased the number of cells exhibiting cytoplasmic immunoreactivity to 12.24 +/- 1.6 (P < 0.05). Ubiquitin immunoreactivity was not observed after GnRH-induced luteal regression. Apoptotic oligonucleosome formation was found after induced luteal regression with both PGF2 alpha and GnRH antagonist, but ubiquitin upregulation only occurred after PGF2 alpha-induced regression. These results indicate that ubiquitin expression is not specific for luteolysis and is not an indicator of luteal apoptosis, but that the polypeptide does play a role in luteal cellular responses to PGF2 alpha.

Messenger ribonucleic acid encoding monocyte chemoattractant protein-1 is expressed by the ovine corpus luteum in response to prostaglandin F2alpha.

To investigate expression of monocyte chemoattractant protein-1 (MCP-1) in the ovine corpus luteum, a partial cDNA was produced by reverse transcription-polymerase chain reaction. This cDNA was 89% identical to that reported for bovine MCP-1 mRNA. In experiment 1, steady-state concentrations of mRNA encoding MCP-1 were measured in pools of luteal tissue collected on Days 3, 6, 9, 12, and 15 of the estrous cycle (estrus = O; n = 4/day). There were no differences in mRNA concentrations for MCP-1 among any of the days studied (p = 0.43). In experiment 2, midluteal-phase corpora lutea were collected from ewes at 0 (untreated), 2, 4, 8, and 16 h after administration of a luteolytic dose of prostaglandin F2alpha (PGF2alpha; n = 4/time point). Concentrations of MCP-1 mRNA were undetectable in untreated controls, were detectable at 2 h post-treatment, had increased 4 and 8 h after administration of PGF2alpha when compared to those at 2 h (p < 0.05), and were decreased 16 h after administration of PGF2alpha when compared to those at 4 h (p < 0.05). In situ hybridization for MCP-1 mRNA combined with immunocytochemical labeling of tissue inhibitor of metalloproteinase-1 (TIMP-1) in large luteal cells was used to determine whether the steroidogenic cells that have PGF2alpha receptors express MCP-1 mRNA in response to PGF2alpha. Messenger RNA encoding MCP-1 and TIMP-1 were not colocalized, indicating that MCP-1 was not expressed by large steroidogenic luteal cells during luteolysis.

Interleukin-1 beta inhibits in vitro pulsatile progesterone secretion and stimulates prostaglandin F2 alpha secretion by micro-retrodialyzed baboon corpus luteum.

Interleukin-1 beta (IL-1 beta) modulates steroidogenesis and prostaglandin (PG) secretion by dispersed luteal cells of some non-primate species. To determine if IL-1 beta affects progesterone (P) and PGF2 alpha secretion by the baboon corpus luteum (CL), we microretrodialyzed the intact CL for 48 h; 12 h media (baseline), 12 h with IL-1 beta (3 IU/h), 12 h media only (post- IL-1 beta) and 12 h with cAMP (5 nmol/h). Four CL from the midluteal phase (LH + 8 days) were studied. P was measured by a sensitive and specific radioimmunoassay and PGF2 alpha by an enzyme immunoassay in the 10-min fractions of retrodialysates collected. P secretions were analyzed for peaks by PC Pulsar (3.0) and total P retrieved/12 h for each experimental segment was calculated. P secretion was pulsatile. Pulses of P declined from 8.2 +/- 1.2/12 h (mean +/- SEM) before to 5.0 +/- 1.2 h after IL-1 beta treatment (P = 0.022), but increased to 10.2 +/- 4.3/12 h with cAMP. Interpulse interval increased significantly from 92 +/- 23 min (baseline) to 137 +/- 31 min (p = 0.025) after IL-1 beta treatment. Total P secreted decreased significantly from 2471 +/- 515 nmol/12 h (baseline) to 1480 +/- 167 nmoles/12 h during IL-1 beta and 788 +/- 85 nmoles/12 h after IL-1 beta (P = 0.015). P was immediately suppressed after starting IL-1 beta in 2 CL but declined only towards the end of treatment in the other 2 CL. PGF2 alpha secretion increased during IL-1 beta with a further increase after IL-1 beta, while P secretion was progressively inhibited. Therefore, IL-1 beta is luteolytic to the primate midluteal phase CL by inhibiting P while simultaneously stimulating PGF2 alpha secretion, demonstrating paracrine-autocrine interaction within the luteal tissue.