Thyroid hormones T3 and T4 regulate human luteinized granulosa cells, counteracting apoptosis and promoting cell survival.

PURPOSE: Fine and balanced regulation of cell proliferation and apoptosis are key to achieve ovarian follicle development from the primordial to the preovulatory stage and therefore assure female reproductive function. While gonadotropins are the major and most recognized regulators of follicle cell growth and function, other factors, both systemic and local, play equally important roles. This work is aimed at evaluating the effects of thyroid hormones (THs) on human granulosa luteinized (hGL) viability. METHODS: Human GL cells derived from assisted reproduction treatments were exposed to T3 or T4. Cell viability was evaluated by MTT assay. Apoptosis was evaluated by the TUNEL assay and active caspase-3 staining. StAR, CYP19A1,Caspase-3, P53 and BAX mRNA were evaluated by real-time PCR. LC3-I/-II, AKT and pAKT were evaluated by western blot. RESULTS: T3 and T4 promoted cell viability in a dose-dependent modality and modulate StAR and CYP19A1 expression. T3 and to a lesser extent T4 mitigated cell death induced by serum starvation by inhibition of caspase-3 activity and expression of P53 and BAX; and attenuate cell death experimentally induced by C2-ceramide. Cell death derived from starvation appeared to be involved in autophagic processes, as the levels of autophagic markers (LC3-II/LC3-I ratio) decreased when starved cells were exposed to T3 and T4. This effect was associated with an increase in pAkt levels. CONCLUSION: From the present study, THs emerge as potent anti-apoptotic agents in hGL cells. This effect is achieved by inhibiting the apoptosis signalling pathway of BAX and caspase-3, while maintaining active the PI3K/AKT pathway.

Gonadotropin regulation of ankyrin-repeat and SOCS-box protein 9 (ASB9) in ovarian follicles and identification of binding partners.

Ankyrin-repeat and SOCS-box protein 9 (ASB9) is a member of the large SOCS-box containing proteins family and acts as the specific substrate recognition component of E3 ubiquitin ligases in the process of ubiquitination and proteasomal degradation. We previously identified ASB9 as a differentially expressed gene in granulosa cells (GC) of bovine ovulatory follicles. This study aimed to further investigate ASB9 mRNA and protein regulation, identify binding partners in GC of bovine ovulatory follicles, and study its function. GC were obtained from small follicles (SF: 2-4 mm), dominant follicles at day 5 of the estrous cycle (DF), and ovulatory follicles, 24 hours following hCG injection (OF). Analyses by RT-PCR showed a 104-fold greater expression of ASB9 in GC of OF than in DF. Steady-state levels of ASB9 in follicular walls (granulosa and theca cells) analyzed at 0, 6, 12, 18 and 24 hours after hCG injection showed a significant induction of ASB9 expression at 12 and 18 hours, reaching a maximum induction of 10.2-fold at 24 hours post-hCG as compared to 0 hour. These results were confirmed in western blot analysis showing strongest ASB9 protein amounts in OF. Yeast two-hybrid screening of OF-cDNAs library resulted in the identification of 10 potential ASB9 binding partners in GC but no interaction was found between ASB9 and creatine kinase B (CKB) in these GC. Functional studies using CRISPR-Cas9 approach revealed that ASB9 inhibition led to increased GC proliferation and modulation of target genes expression. Overall, these results support a physiologically relevant role of ASB9 in the ovulatory follicle by targeting specific proteins likely for degradation, contributing to reduced GC proliferation, and could be involved in the final GC differentiation into luteal cells.

Embryotrophic effect of a short-term embryo coculture with bovine luteal cells.

The coculture with somatic cells is an alternative to improve suboptimal in vitro culture (IVC) conditions and promote embryo development. Several cell types have been used for this purpose, but there is no information about using luteal cells in short-term coculture with embryos. Consequently, this study aimed to assess the effect of a short-term coculture of early bovine embryos-luteal cells on the in vitro development and embryo quality. Presumptive embryos were cultured from day 0 to day 2 in medium alone (control) or cocultured with bovine luteal cells (BLC-1). Then, embryos from both groups were cultured in medium alone from day 2 to day 8. The development rates on day 8 were compared between groups. The level of reactive oxygen species (ROS) and proliferation rates were evaluated in day 2 embryos and late apoptosis and proliferation rates were determined in day 7 blastocysts. Our results showed that the coculture with bovine luteal cells increased the blastocyst rate compared to the control (50.4% vs. 29.8%; P < 0.01), but there were no differences in the cleavage rates on day 2. The rate of stage 6 blastocysts was higher in the coculture (37.3% vs. 23.8% control; P < 0.01), without differences in the expansion and hatching rates compared to the control. The ROS level in day 2 embryos was higher in the coculture than the control (82 vs. 57.1; P < 0.05), and the cell proliferation rate was higher in the coculture (48% vs. 13% control; P < 0.01), without differences in the mean number of cells between groups. In day 7 blastocysts, the apoptosis rate decreased in the coculture with bovine luteal cells from day 0 to day 2 (4.1% vs. 10.9% control; P < 0.01), whereas the cell proliferation rate and the mean number of cells did not differ between groups. This is the first report of a short-term coculture of in vitro produced embryos and bovine luteal cells. Our model could be an alternative to increase the efficiency of the in vitro production of embryos in cattle.

The direct and indirect effects of kisspeptin-54 on granulosa lutein cell function.

STUDY QUESTION: What are the in vivo and in vitro actions of kisspeptin-54 on the expression of genes involved in ovarian reproductive function, steroidogenesis and ovarian hyperstimulation syndrome (OHSS) in granulosa lutein (GL) cells when compared with traditional triggers of oocyte maturation? SUMMARY ANSWER: The use of kisspeptin-54 as an oocyte maturation trigger augmented expression of genes involved in ovarian steroidogenesis in human GL cells including, FSH receptor (FSHR), LH/hCG receptor (LHCGR), steroid acute regulatory protein (STAR), aromatase, estrogen receptors alpha and beta (ESR1, ESR2), 3-beta-hydroxysteroid dehydrogenase type 2 (3BHSD2) and inhibin A (INHBA), when compared to traditional maturation triggers, but did not alter markers of OHSS. WHAT IS KNOWN ALREADY: hCG is the most widely used trigger of oocyte maturation, but is associated with an increased risk of OHSS. The use of GnRH agonists to trigger oocyte maturation is a safer alternative to hCG. More recently, kisspeptin-54 has emerged as a novel therapeutic option that safely triggers oocyte maturation even in women at high risk of OHSS. Kisspeptin indirectly stimulates gonadotropin secretion by acting on hypothalamic GnRH neurons. Kisspeptin and its receptor are also expressed in the human ovary, but there is limited data on the direct action of kisspeptin on the ovary. STUDY DESIGN SIZE, DURATION: Forty-eight women undergoing IVF treatment for infertility consented to kisspeptin-54 triggering and/or granulosa cell collection and were included in the study. Twelve women received hCG, 12 received GnRH agonist and 24 received kisspeptin-54 to trigger oocyte maturation. In the kisspeptin-54 group, 12 received one injection of kisseptin-54 (9.6 nmol/kg) and 12 received two injections of kisspeptin-54 at a 10 h interval (9.6 nmol/kg × 2). PARTICIPANTS/MATERIALS, SETTING, METHODS: Follicular fluid was aspirated and pooled from follicles during the retrieval of oocytes for IVF/ICSI. GL cells were isolated and either RNA extracted immediately or cultured in vitro ± kisspeptin or hCG. MAIN RESULTS AND THE ROLE OF CHANCE: GL cells from women who had received kisspeptin-54 had a 14-fold and 8-fold higher gene expression of FSHR and a 2-fold (ns) and 2.5-fold (P < 0.05) higher expression of LHCGR than GL cells from women who had received hCG or GnRH agonist, respectively. CYP19A1 expression was 3.6-fold (P < 0.05) and 4.5-fold (P < 0.05) higher, STAR expression was 3.4-fold (P < 0.01) and 1.8-fold (P < 0.05) higher, HSD3B2 expression was 7.5- (P < 0.01) and 2.5-fold higher (P < 0.05), INHBA was 2.5-fold (P < 0.01) and 2.5-fold (P < 0.01) higher in GL cells from women who had received kisspeptin-54 than hCG or GnRHa, respectively. ESR1 (P < 0.05) and ESR2 (P < 0.05) both showed 3-fold higher expression in cells from kisspeptin treated than GnRHa treated women. Markers of vascular permeability and oocyte growth factors were unchanged (VEGFA, SERPINF1, CDH5, amphiregulin, epiregulin). Gene expression of kisspeptin receptor was unchanged. Whereas treating GL cells in vitro with hCG induced steroidogenic gene expression, kisspeptin-54 had no significant direct effects on either OHSS genes or steroidogenic genes. LIMITATIONS REASONS FOR CAUTION: Most women in the study had PCOS, which may limit applicability to other patient groups. For the analysis of the in vitro effects of kisspeptin-54, it is important to note that GL cells had already been exposed in vivo to an alternate maturation trigger. WIDER IMPLICATIONS OF THE FINDINGS: The profile of serum gonadotropins seen with kisspeptin administration compared to other triggers more closely resemble that of the natural cycle as compared with hCG. Thus, kisspeptin could potentially permit an ovarian environment augmented for steroidogenesis, in particular progesterone synthesis, which is required for embryo implantation. STUDY FUNDING/COMPETING INTEREST(S): Dr Owens is supported by an Imperial College London PhD Scholarship. Dr Abbara is supported by an National Institute of Health Research Academic Clinical Lectureship. The authors do not have any conflict of interest to declare. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov NCT01667406.

AG 205, a progesterone receptor membrane component 1 antagonist, ablates progesterone's ability to block oxidative stress-induced apoptosis of human granulosa/luteal cells†.

The present studies were designed to determine whether progesterone (P4)-progesterone receptor membrane component 1 (PGRMC1) signaling is able to attenuate the apoptotic effects of oxidative stress induced by hydrogen peroxide (H2O2). To achieve this goal, freshly isolated human granulosa/luteal cells were maintained in culture. After several passages, the cells were treated with H2O2, which induced apoptosis within 2.5 h, while simultaneous treatment with P4 attenuated the apoptotic action of H2O2. AG 205, a PGRMC1 antagonist, eliminated P4's ability to prevent H2O2-induced apoptosis. AG 205 neither affected PGRMC1's cytoplasmic localization nor its interaction with PGRMC2, but appeared to reduce its presence within the nucleus. AG 205 also (1) increased the monomeric and decreased the higher molecular weight forms of PGRMC1 (i.e., dimers/oligomers) and (2) altered the expression of several genes involved in apoptosis. The most dramatic change was an approximate 8-fold increase in Harakiri (Hrk) mRNA. However, AG 205 did not induce apoptosis in the absence of H2O2. Taken together, these observations suggest that the higher molecular weight forms of PGRMC1 likely account in part for PGRMC1's ability to suppress the expression of Hrk. Harakiri is a BH-3 only member of the B-cell lymphoma 2 (BCL2) family that promotes apoptosis by binding to and antagonizing the antiapoptotic action of BCL2- and BCL2-like proteins. It is likely then that PGRMC1's ability to suppress Hrk is part of the mechanism through which P4-PGRMC1 signaling preserves the viability of human granulosa/luteal cells.

Expression and localization of angiopoietin family in corpus luteum during different stages of oestrous cycle and modulatory role of angiopoietins on steroidogenesis, angiogenesis and survivability of cultured buffalo luteal cells.

The objective of this study was to document the expression and localization of angiopoietin (ANGPT) family members comprising of angiopoietin (ANGPT1 and ANGPT2), and their receptors (Tie1 and Tie2) in buffalo corpus luteum (CL) obtained from different stages of the oestrous cycle, and the modulatory role of ANGPT1 and ANGPT2 alone or in combinations on progesterone (P4 ) secretion and mRNA expression of phosphotidylinositide-3kinase-protein kinase B (PI3K-AKT), phosphoinositide-dependent kinase (PDK), protein kinase B (AKT), Bcl2 associated death promoter (BAD), caspase 3 and von willebrand factor (vWF) in luteal cells obtained from midluteal phase (MLP) of oestrous cycle in buffalo. Real-time RT-PCR (qPCR), Western blot and immunohistochemistry were applied to investigate mRNA expression, protein expression and localization of examined factors whereas, the P4 secretion was assessed by RIA. The mRNA and protein expression of ANGPT1 and Tie2 was maximum (p < .05) in mid luteal phase (MLP) of oestrous cycle. The ANGPT2 mRNA and protein expression was maximum (p < .05) in early luteal phase, decreased in MLP and again increased in late luteal phase of oestrous cycle. ANGPT family members were localized in luteal cells and endothelial cells with a stage specific immunoreactivity. P4 secretion was highest (p < .05) with 100 ng/ml at 72 hr when luteal cells were treated with either protein alone. The mRNA expression of PDK, AKT and vWF was highest (p < .05) and BAD along with caspase 3 were lowest (p < .05) at 100 ng/ml at 72 hr of incubation period, when cultured luteal cells were treated with either protein alone or in combination. To conclude, our study explores the steroidogenic potential of angiopoietins to promote P4 secretion, luteal cell survival and angiogenesis through an autocrine and paracrine actions 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), 3ß-hydroxysteroid dehydrogenase (3ß-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.

Expression and contribution of the HIF-1α/VEGF signaling pathway to luteal development and function in pregnant rats.

Vascular endothelial growth factor (VEGF) is vital in normal and abnormal angiogenesis in the ovary, particularly during the early development of the corpus luteum in the ovary. However, the molecular regulation of the expression VEGF during luteal development in vivo remains to be fully elucidated. As the expression of VEGF is mediated by hypoxia­inducible factor (HIF)­1α in luteal cells cultured in vitro, determined in our previous study, the present study was performed to confirm the hypothesis that HIF­1α is induced and then regulates the expression of VEGF and VEGF­dependent luteal development/function in vivo. This was investigated using a pregnant rat model treated with a small­molecule inhibitor of HIF­1α, echinomycin (Ech). The development of the corpus luteum in the pregnant rat ovary was identified via performing assays of the serum progesterone, testosterone and estradiol concentrations by radioimmunoassay, accompanied with determination of the changes in the expression levels of HIF­1α and VEGF by reverse transcription­quantitative polymerase chain reaction at different days of the developmental process. On day 5, serum progesterone levels were markedly increased, whereas serum levels of testosterone and estradiol did not change significantly. On day 17, the highest level of serum progesterone was observed, however, this was not the case for testosterone and estradiol. Further analysis of the expression levels of HIF­1α and VEGF revealed that their changes were consistent with the changes in serum levels of progesterone, which occurred in the development of the corpus luteum in the ovaries of pregnant rats. Further investigation demonstrated that Ech inhibited luteal development through inhibiting the expression of VEGF, mediated by HIF­1α, and subsequent luteal function, which was determined by detecting changes in serum progesterone on days 8 and 14. Taken together, these results demonstrated that HIF­1α­mediated expression of VEGF may be one of the important mechanisms regulating ovarian luteal development in mammals in vivo, which may provide novel strategies in treatment for fertility control and for certain types of ovarian dysfunction, including polycystic ovarian syndrome, ovarian hyperstimulation syndrome and ovarian neoplasia.

Effects of recombinant activins on steroidogenesis in human granulosa-lutein cells.

CONTEXT: Exerting a broad range of biological effects in various tissues, activins are homo- or heterodimers of activin/inhibin ß-subunits (ßA, ßB, ßC, and ßE in humans). Although activins A (ßAßA), B (ßBßB), AB (ßAßB), and AC (ßAßC) have been demonstrated in the female reproductive system, little is known about their individual functions in the ovary. OBJECTIVE: To investigate the biological roles and activities of activins in regulating steroidogenesis in human granulosa cells. DESIGN: Human granulosa-lutein cells obtained from 32 patients undergoing in vitro fertilization were used to investigate the effects of activin A, B, AB, and AC on the expression of steroidogenic enzymes and steroid production. SETTING: An academic research center. MAIN OUTCOME MEASURES: mRNA and protein levels were examined by reverse transcription quantitative real-time PCR and Western blot analysis, respectively. The production of estradiol and progesterone was measured by enzyme immunoassay. RESULTS: P450 aromatase, FSH receptor, and estradiol levels were increased, whereas steroidogenic acute regulatory protein (StAR), LH receptor, and progesterone levels were decreased after treatment with activin A, B, and AB, but not activin AC. FSH or LH induced the production of aromatase/estradiol and StAR/progesterone; however, pretreatment with activin A, B, or AB enhanced the effects of gonadotropins on aromatase/estradiol, but suppressed their effects on StAR/progesterone. Treatment with activin A, B, or AB induced the phosphorylation of SMA- and MAD-related proteins (SMAD2 and 3), whereas activin AC had no such effects. Furthermore, co-culture of activin AC (1-100 ng/mL) with activin A (25 ng/mL) did not alter the effects of activin A on P450 aromatase or StAR mRNA levels. CONCLUSION: Activin A, B, and AB have similar effects on steroidogenesis in human granulosa cells. In contrast, activin AC is not biologically active and does not act as a competitive antagonist.

Stimulatory effect of vascular endothelial growth factor on progesterone production and survivability of cultured bubaline luteal cells.

The objectives of the present study were to investigate the effects of vascular endothelial growth factor (VEGF) on progesterone (P4) synthesis in cultured luteal cells from different stages of the estrous cycle and on expression of steroidogenic acute regulatory protein (STARD1), cytochrome P450 cholesterol side chain cleavage (CYP11A1) and 3ß-hydroxysteroid dehydrogenase (HSD3B), antiapoptotic gene PCNA, and proapoptotic gene BAX in luteal cells obtained from mid-luteal phase (MLP) of estrous cycle in buffalo. Corpus luteum samples from the early luteal phase (ELP; day 1st-4th; n=4), MLP (day 5th-10th; n=4), and the late luteal phase (LLP; day 11th-16th; n=4) of oestrous cycle were obtained from a slaughterhouse. Luteal cell cultures were treated with VEGF (0, 1, 10 and 100 ng/ml) for 24, 48 and 72h. Progesterone was assessed by RIA, while mRNA expression was determined by quantitative real-time PCR (qRT-PCR). Results indicated a dose- and time-dependent stimulatory effect of VEGF on P4 synthesis and expression of steroidogenic enzymes. Moreover, VEGF treatment led to an increase in PCNA expression and decrease in BAX expression. In summary, these findings suggest that VEGF acts locally in the bubaline CL to modulate steroid hormone synthesis and cell survivability, which indicates that this factor has an important role as a regulator of CL development and function in buffalo.

Non-canonical progesterone signaling in granulosa cell function.

It has been known for over 3 decades that progesterone (P4) suppresses follicle growth. It has been assumed that P4 acts directly on granulosa cells of developing follicles to slow their development, as P4 inhibits both mitosis and apoptosis of cultured granulosa cells. However, granulosa cells of developing follicles of mice, rats, monkeys, and humans do not express the A or B isoform of the classic nuclear receptor for P4 (PGR). By contrast, these granulosa cells express other P4 binding proteins, one of which is referred to as PGR membrane component 1 (PGRMC1). PGRMC1 specifically binds P4 with high affinity and mediates P4's anti-mitotic and anti-apoptotic action as evidenced by the lack of these P4-dependent effects in PGRMC1-depleted cells. In addition, mice in which PGRMC1 is conditionally depleted in granulosa cells show diminished follicle development. While the mechanism through which P4 activation of PGRMC1 affects granulosa cell function is not well defined, it appears that PGRMC1 controls granulosa cell function in part by regulating gene expression in T-cell-specific transcription factor/lymphoid enhancer factor-dependent manner. Clinically, altered PGRMC1 expression has been correlated with premature ovarian failure/insufficiency, polycystic ovarian syndrome, and infertility. These collective studies provide strong evidence that PGRMC1 functions as a receptor for P4 in granulosa cells and that altered expression results in compromised reproductive capacity. Ongoing studies seek to define the components of the signal transduction cascade through which P4 activation of PGRMC1 results in the regulation of granulosa cell function.

Influence of lysophosphatidic acid on nitric oxide-induced luteolysis in steroidogenic luteal cells in cows.

Lysophosphatidic acid (LPA) together with its active G protein-coupled receptors are present in the corpus luteum (CL) of the cow. Under in vivo conditions, LPA stimulated P4 and PGE2 secretion during the luteal phase of the estrous cycle in heifers. Furthermore, LPA maintained P4 synthesis and actions in the bovine CL in vitro. However, the effect of this phospholipid on nitric oxide (NO)-induced functional and structural luteolysis has not been investigated. The aim of the present work was to determine the effects of LPA on 1) NO-induced functional luteolysis, 2) NO-dependent PG synthesis, and 3) NO-induced structural luteolysis in cultured steroidogenic luteal cells. We documented that LPA reversed the inhibitory effect of NONOate, an NO donor, on P4 synthesis and PGE2/PGF2alpha ratio in cultured steroidogenic luteal cells. Additionally, LPA inhibited NO-induced apoptosis in cultured steroidogenic luteal cells via abrogation of the NO-dependent stimulatory influence on proapoptotic TNFalpha/TNFR1 and Fas/FasL expression, Caspase 3 activity, and the Bax/Bcl2 ratio during luteal regression in the bovine CL. In conclusion, this study proves that in the presence of LPA, NO cannot induce luteolytic capacity acquisition, leading to functional and structural luteolysis of bovine luteal cells.

Mathematical analysis of a model for the growth of the bovine corpus luteum.

The corpus luteum (CL) is an ovarian tissue that grows in the wound space created by follicular rupture. It produces the progesterone needed in the uterus to maintain pregnancy. Rapid growth of the CL and progesterone transport to the uterus require angiogenesis, the creation of new blood vessels from pre-existing ones, a process which is regulated by proteins that include fibroblast growth factor 2 (FGF2). In this paper we develop a system of time-dependent ordinary differential equations to model CL growth. The dependent variables represent FGF2, endothelial cells (ECs), luteal cells, and stromal cells (like pericytes), by assuming that the CL volume is a continuum of the three cell types. We assume that if the CL volume exceeds that of the ovulated follicle, then growth is inhibited. This threshold volume partitions the system dynamics into two regimes, so that the model may be classified as a Filippov (piecewise smooth) system. We show that normal CL growth requires an appropriate balance between the growth rates of luteal and stromal cells. We investigate how angiogenesis influences CL growth by considering how the system dynamics depend on the dimensionless EC proliferation rate, ρ5. We find that weak (low ρ5) or strong (high ρ5) angiogenesis leads to 'pathological' CL growth, since the loss of CL constituents compromises progesterone production or delivery. However, for intermediate values of ρ5, normal CL growth is predicted. The implications of these results for cow fertility are also discussed. For example, inadequate angiogenesis has been linked to infertility in dairy cows.

Influence of prostaglandin F2α analogues on the secretory function of bovine luteal cells and ovarian arterial contractility in vitro.

Although prostaglandin (PG) F2α analogues are routinely used for oestrus synchronisation in cattle, their effects on the function of the bovine corpus luteum (CL), and on ovarian arterial contractility, may not reflect the physiological effects of endogenous PGF2α. In the first of two related experiments, the effects of different analogues of PGF2α (aPGF2α) on the secretory function and apoptosis of cultured bovine cells of the CL were assessed. Enzymatically-isolated bovine luteal cells (from between days 8 and 12 of the oestrous cycle), were stimulated for 24h with naturally-occurring PGF2α or aPGF2α (dinoprost, cloprostenol or luprostiol). Secretion of progesterone (P4) was determined and cellular [Ca(2+)]i mobilisation, as well as cell viability and apoptosis were measured. Naturally-occurring PGF2α and dinoprost stimulated P4 secretion (P<0.05), whereas cloprostenol and luprostiol did not influence P4 synthesis. The greatest cytotoxic and pro-apoptotic effects were observed in the luprostiol-treated cells, at 37.3% and 202%, respectively (P<0.001). The greatest effect on [Ca(2+)]i mobilisation in luteal cells was observed post-luprostiol treatment (200%; P<0.001). In a second experiment, the influence of naturally-occurring PGF2α and aPGF2α on ovarian arterial contraction in vitro, were examined. No differences in the effects of dinoprost or naturally-occurring PGF2α were found across the studied parameters. The effects of cloprostenol and luprostiol on luteal cell death, in addition to their effects on ovarian arterial contractility, were much greater than those produced by treatment with naturally-occurring PGF2α.

Ovarian expression, localization, and function of tissue inhibitor of metalloproteinase 3 (TIMP3) during the periovulatory period of the human menstrual cycle.

Ovulation involves reorganization of the extracellular matrix of the follicle. This study examines the expression, localization, and potential function of the tissue inhibitor of metalloproteinase 3 (TIMP3) during ovulation in women. The dominant follicle of the menstrual cycle was collected at specified times throughout the ovulatory process: pre-, early, late, and postovulatory. For quantitative studies, the follicle was bisected; granulosa and theca cells were separated and collected. For immunohistochemistry (IHC), the intact follicle was embedded and TIMP3 was localized. Additionally, granulosa cells were collected from women undergoing in vitro fertilization and treated with increasing concentrations of recombinant TIMP3, and cell viability was assessed. Real-time PCR for TIMP3 mRNA revealed an increase in TIMP3 mRNA expression in granulosa cells from the early to the late ovulatory stage. Thecal TIMP3 mRNA expression was constitutive across the periovulatory period. TIMP3 protein was localized by IHC to the granulosa and theca cell layers in pre-, early, and late ovulatory follicles as well as to the vascular bed. The staining was most intense in the granulosa and theca cells in the late ovulatory group. Treatment of human granulosa-lutein cells with exogenous recombinant TIMP3 for 24 h decreased cell viability by 60%. Using human follicles collected throughout the periovulatory period of the menstrual cycle, we have demonstrated that TIMP3 mRNA expression increases and that TIMP3 protein is in the appropriate cellular layers to regulate proteolytic remodeling as the follicle progresses toward ovulation. In addition, we have shown that elevated levels of TIMP3 lead to decreased cell viability.

Isolation and characterization of luteal cells in buffalo (Bubalus bubalis).

The objective of the study was to evaluate the morphological and functional characteristics of luteal cells isolated from buffalo ovary. Luteal cells exhibited columnar morphology and contact inhibition at the stage of confluence. Protein concentrations increased linearly from Day 3 to Day 7 of culture. DNA concentrations increased from Day 3 to Day 5 and then declined to Day 7 of culture. PGF2alpha concentrations decreased progressively from Day 3 to Day 7 of culture. It was concluded that buffalo luteal cells could serve as an excellent model for studying the specific role of PGF2alpha in maternal recognition of pregnancy and implantation.

Cellular and functional characterization of buffalo (Bubalus bubalis) corpus luteum during the estrous cycle and pregnancy.

In the present paper, cellular composition of buffalo corpus luteum (CL) with its functional characterization based on 3ß-HSD and progesterone secretory ability at different stages of estrous cycle and pregnancy was studied. Buffalo uteri along with ovaries bearing CL were collected from the local slaughter house. These were classified into different stages of estrous cycle (Stage I, II, III and IV) and pregnancy (Stage I, II and III) based on morphological appearance of CL, surface follicles on the ovary and crown rump length of conceptus. Luteal cell population, progesterone content and steroidogenic properties were studied by dispersion of luteal cells using collagenase type I enzyme, RIA and 3ß-HSD activity, respectively. Large luteal cells (LLC) appeared as polyhedral or spherical in shape with a centrally placed large round nucleus and an abundance of cytoplasmic lipid droplets. However, small luteal cells (SLC) appeared to be spindle shaped with an eccentrically placed irregular nucleus and there was paucity of cytoplasmic lipid droplets. The size of SLC (range 12-23µm) and LLC (range 25-55µm) increased (P<0.01) with the advancement of stage of estrous cycle and pregnancy. The mean progesterone concentration per gram and per CL increased (P<0.01) from Stage I to III of estrous cycle with maximum concentration at Stage III of estrous cycle and pregnancy. The progesterone concentration decreased at Stage IV (day 17-20) of estrous cycle coinciding with CL regression. Total luteal cell number (LLC and SLC) also increased (P<0.01) from Stage I to III of estrous cycle and decreased (P<0.05), thereafter, at Stage IV indicating degeneration of luteal cells and regression of the CL. Total luteal cell population during pregnancy also increased (P<0.01) from Stage I to II and thereafter decreased (P>0.05) indicating cessation of mitosis. Increased (P<0.05) large luteal cell numbers from Stage I to III of estrous cycle and pregnancy coincided with the increased progesterone secretion and 3ß-HSD activity of CL. Thus, proportionate increases of large compared with small luteal cells were primarily responsible for increased progesterone secretion during the advanced stages of the estrous cycle and pregnancy. Total luteal cells and progesterone content per CL during the mid-luteal stage in buffalo as observed in the present study seem to be less than with cattle suggesting inherent luteal deficiency.

Progesterone vaginal ring versus vaginal gel for luteal support with in vitro fertilization: a randomized comparative study.

OBJECTIVE: To compare the efficacy and safety of luteal phase support in IVF with a progesterone (P) vaginal ring or gel (VR or VG). DESIGN: Prospective, randomized, single-blind, multicenter, phase III clinical trial (ClinicalTrials.gov identifier: NCT00615251). SETTING: Nineteen private and three academic high-volume U.S. IVF centers. PATIENT(S): One thousand two hundred ninety-seven infertile patients were randomized to a weekly P VR (n = 646) or a daily P 8% VG (n = 651). INTERVENTION(S): IVF was performed per site-specific protocols. The day after egg retrieval, patients were randomized and began VR or VG therapy, which continued for up to 10 weeks' gestation. MAIN OUTCOME MEASURE(S): Clinical pregnancy rates at 8 and 12 weeks of pregnancy; rates of biochemical pregnancy, live birth, spontaneous abortion, ectopic pregnancy, and cycle cancellation; and safety and tolerability were secondary measures. RESULT(S): Clinical pregnancy rates at 8 and 12 weeks were high and comparable between groups: 48.0% for VR and 47.2% for VG at week 8 and 46.4% (VR) and 45.2% (VG) at week 12. Live-birth rates were 45% (VR) and 43% (VG). Adverse event profiles were similar between groups. CONCLUSION(S): The weekly P VR provided similar pregnancy rates to the daily VG, with no major differences in safety.

Human chorionic gonadotropin controls luteal vascular permeability via vascular endothelial growth factor by down-regulation of a cascade of adhesion proteins.

OBJECTIVE: To study the functional interactions of junctional proteins acting as regulators of vascular permeability in the human corpus luteum. We investigated the role of vascular endothelial (VE)-cadherin, nectin 2, and claudin 5 as controllers of vascular endothelial cell permeability. DESIGN: Performing immunohistochemical dual staining, we colocalized the above-mentioned proteins in the human corpus luteum. SETTING: Not applicable. PATIENT(S): Not applicable. INTERVENTION(S): Not applicable. MAIN OUTCOME MEASURE(S): Using a granulosa-endothelial coculture system, we revealed that hCG-treatment down-regulates VE-cadherin, nectin 2, and claudin 5 in endothelial cells via vascular endothelial growth factor (VEGFA). RESULT(S): Furthermore, the interaction of VE-cadherin, nectin 2, and claudin 5 was investigated by silencing these proteins that perform siRNA knockdown. Interestingly, knockdown of VE-cadherin and claudin 5 induced a decrease of the respective other protein. This down-regulation was associated with changed rates of vascular permeability: hCG induced a VEGFA-dependent down-regulation of VE-cadherin, nectin 2, and claudin 5, which increased the endothelial permeability in the coculture system. Furthermore, knockdown of VE-cadherin, nectin-2, and claudin 5 also resulted in a consecutive increase of endothelial permeability for each different protein. CONCLUSION(S): These results demonstrate for the first time that VE-cadherin, nectin 2, and claudin 5 are involved in the regulation of vascular permeability in a mutually interacting manner, which indicates their prominent role for the functionality of the human corpus luteum.

Role of the cell cycle in regression of the corpus luteum.

The corpus luteum contains differentiated steroidogenic cells that have exited the cell cycle of proliferation. In some tissues, deletion of quiescent, differentiated cells by apoptosis in response to injury or pathology is preceded by reentry into the cell cycle. We tested whether luteal cells reenter the cell cycle during the physiological process of luteolysis. Ovaries were obtained after injection of cows with a luteolytic dose of prostaglandin F(2)(α) (PGF). In luteal sections, cells co-staining for markers of cell proliferation (MKI67) and apoptosis (cPARP1) increased 24  h after PGF, indicating that cells that reenter the cell cycle undergo apoptosis. The percent of steroidogenic cells (CYP11A1-positive) co-staining for MKI67 increased after PGF, while co-staining of non-steroidogenic cells did not change. Dispersed luteal cells were stained with Nile Red to distinguish lipid-rich steroidogenic cells from nonsteroidogenic cells and co-stained for DNA. Flow cytometry showed that the percent of steroidogenic cells progressing through the cell cycle and undergoing apoptosis increased after PGF. Culturing luteal cells induced reentry of steroidogenic cells into the cell cycle, providing a model to test the influence of the cell cycle on susceptibility to apoptosis. Blocking cells early in the cell cycle using inhibitors reduced cell death in response to treatment with the apoptosis-inducing protein, Fas ligand (FASL). Progesterone treatment reduced progression through the cell cycle and decreased FASL-induced apoptosis. In summary, steroidogenic cells reenter the cell cycle upon induction of luteal regression. While quiescent cells are resistant to apoptosis, entry into the cell cycle promotes susceptibility to apoptosis.