Corpus Luteum Tissue Research Articles

Oxylipin concentrations in bovine corpora lutea during maternal recognition of pregnancy

The objective was to determine the effects of pregnancy status on oxylipin profiles and eicosanoid metabolizing enzymes and in corpora lutea (CL) or endometrial (caruncle; CAR and intercaruncle; IC) tissues. Angus crossed cattle were synchronized with the CO-Synch protocol and artificially inseminated (AI). Sixteen days after AI, cattle were euthanized, and reproductive tracts collected from 6 non-pregnant and 6 pregnant cows. Oxylipin profiles and concentrations of progesterone (P4) were obtained from CL tissues. The activity of cytochrome P450 1A (CYP1A) and UDP-glucuronosyltransferase (UGT) enzymes were determined using specific luminogenic substrates. Data were analyzed using the MIXED procedure of SAS, and the model included pregnancy status. Corpora lutea of pregnant cattle contained greater (P < 0.05) concentrations of 9,10-DiHODE, 15,16-DiHODE, and 9,10-DiHOME. These oxylipins have been observed to increase cellular proliferation and vasodilation. Activity of CYP1A in the CL and UGT in CAR and IC was not different (P > 0.05) between pregnant and non-pregnant cattle. In the CL, activity of UGT was decreased (P < 0.05) in pregnant vs. non-pregnant cattle. The decrease in CL UGT activity during pregnancy indicates alterations in local hormone metabolism, while no differences in CL weight nor amount of P4 in CL were different between pregnant and non-pregnant cattle. Moreover, the increase in specific concentrations of oxylipins in the CL may indicate a novel pathway of steroid and eicosanoid metabolism during maternal recognition of pregnancy.

219 Use of a genetically-engineered swine line to elucidate the role of GnRH-II and its receptor in gilts

Abstract The second form of GnRH (GnRH-II) and its receptor (GnRHR-II) are produced in only one livestock species, the pig. Paradoxically, their interaction does not stimulate gonadotropin secretion. Instead, both have been implicated in autocrine/paracrine regulation of steroidogenesis. To elucidate their role in ovarian function, our laboratory generated transgenic swine with ubiquitous knockdown (KD) of GnRHR-II. Blood samples were collected from GnRHR-II KD (n = 8) and littermate control (n = 7) gilts at the onset of estrus (follicular) and 10 d later (luteal). Serum samples were subjected to HPLC-MS/MS to quantify concentrations of 16 steroid hormones. At euthanasia, ovarian weight, ovulation rate and weight of each excised corpus luteum (CL) were recorded; HPLC-MS/MS was also performed on CL tissue. A line (GnRHR-II KD versus control) x phase (follicular versus luteal) interaction was detected for serum progesterone concentrations; levels were reduced in transgenic compared with control gilts during the luteal phase (P = 0.0329). A tendency for a line effect was observed for 11-deoxycorticosterone and 11-deoxycortisol; transgenic females tended to produce less of these corticosteroids (P &lt; 0.10). A phase effect was detected for cortisone, 11-deoxycortisol, cortisol, corticosterone, androstenedione, androsterone, testosterone, estrone and 17β-estradiol (P &lt; 0.05); concentrations were greater in follicular versus luteal samples (P &lt; 0.05). Conversely, 17α-hydroxyprogesterone concentrations were elevated in luteal samples (P &lt; 0.05). Ovarian weight did not differ between lines, although ovulation rate was reduced in GnRHR-II KD versus control gilts (P = 0.0123). However, average CL weight was greater in GnRHR-II KD compared with control females (P &lt; 0.0001); therefore, total CL weight tended to be reduced in transgenic gilts (P = 0.0958). In tissue samples, concentrations of progesterone and estrone tended to be reduced in transgenics females (P ≤ 0.10). Ultimately, these data suggest that GnRH-II and its receptor may help regulate ovulation rate, CL development and progesterone production in gilts. Supported by USDA/NIFA AFRI-ELI predoctoral fellowship (2017-67011-26036; ATD) and AFRI (2017-67015-26508; BRW) funds.

Prostaglandins in Superovulation Induced Bovine Follicles During the Preovulatory Period and Early Corpus Luteum.

The aim of this study was to characterize the regulation pattern of prostaglandin family members namely prostaglandin F2alpha (PTGF), prostaglandin E2 (PTGE), their receptors (PTGFR, PTGER2, PTGER4), cyclooxygenase 2 (COX-2), PTGF synthase (PTGFS), and PTGE synthase (PTGES) in the bovine follicles during preovulatory period and early corpus luteum (CL). Ovaries containing preovulatory follicles or CL were collected by transvaginal ovariectomy (n = 5 cows/group), and the follicles were classified: (I) before GnRH treatment; (II) 4 h after GnRH; (III) 10 h after GnRH; (IV) 20 h after GnRH; (V) 25 h after GnRH, and (VI) 60 h after GnRH (early CL). In these samples, the concentrations of progesterone (P4), estradiol (E2), PTGF and PTGE were investigated in the follicular fluid (FF) by validated EIA. Relative mRNA abundance of genes encoding for prostaglandin receptors (PTGFR, PTGER2, PTGER4), COX-2, PTGFS and PTGES were quantified by RT-qPCR. The localization of COX-2 and PTGES were investigated by established immunohistochemistry in fixed follicular and CL tissue samples. The high E2 concentration in the FF of the follicle group before GnRH treatment (495.8 ng/ml) and during luteinizing hormone (LH) surge (4 h after GnRH, 574.36 ng/ml), is followed by a significant (P<0.05) downregulation afterwards with the lowest level during ovulation (25 h after GnRH, 53.11 ng/ml). In contrast the concentration of P4 was very low before LH surge (50.64 mg/ml) followed by a significant upregulation (P < 0.05) during ovulation (537.18 ng/ml). The mRNA expression of COX-2 increased significantely (P < 0.05) 4 h after GnRH and again 20 h after GnRH, followed by a significant decrease (P < 0.05) after ovulation (early CL). The mRNA of PTGFS in follicles before GnRH was high followed by a continuous and significant downregulation (P < 0.05) afterwards. In contrast, PTGES mRNA abundance increased significantely (P < 0.05) in follicles 20 h after GnRH treatment and remained high afterwards. The mRNA abundance of PTGFR, PTGER2, and PTGER4 in follicles before GnRH was high, followed by a continuous and significant down regulation afterwards and significant increase (P < 0.05) only after ovulation (early CL). The low concentration of PTGF (0.04 ng/ml) and PTGE (0.15 ng/ml) in FF before GnRH, increased continuously in follicle groups before ovulation and displayed a further significant and dramatic increase (P < 0.05) around ovulation (101.01 ng/ml, respectively, 484.21 ng/ml). Immunohistochemically, the granulosa cells showed an intensive signal for COX-2 and PTGES in follicles during preovulation and in granulosa-luteal cells of the early CL. In conclusion, our results indicate that the examined bovine prostaglandin family members are involved in the local mechanisms regulating final follicle maturation and ovulation during the folliculo-luteal transition and CL formation.

MicroRNA-related markers associated with corpus luteum tropism in buffalo (Bubalus bubalis)

The study was undertaken to decipher the microRNA (miRNA) related markers associated with corpus luteum (CL) tropism in buffalo. The data obtained from deep sequencing of CL tissue from different physiological stages was mined in silico for the identification of miRNA-related markers (SSR & SNP). From the present study, 5 annotated and 176 unannotated miRNA were deduced while comparing with Bos taurus genome. In addition, 4 SSRs and 9 SNPs were deduced from the miRNA sequences. These SSRs were on the genes viz. Eukaryotic translation initiation factor 1-like, myocyte enhancer factor 2A, beta casein, T cell receptor gamma cluster 1. The SNP positions on genes viz. PYGO1 (Pygopus family PHD finger 1), LOC100337244 (Multidrug resistance-associated protein 4), FTH1 (Ferritin heavy chain 1), LOC788634 (BOLA class I histocompatibility antigen), PLXND1 (Plexin D1) and UBC (Ubiquitin C) show that these genes play critical role in CL tropism during estrous cycle in buffalo.

Possible roles of local oviductal estradiol-17β in luteal formation phase on the function of bovine oviductal epithelium.

Oviducts play an important role in the reproductive process, such as in gamete transport, fertilization, and early embryonic development. However, the regulation of oviductal function during luteal formation phase (3−5 days post-ovulation), which is a crucial phase for early embryonic development, remains poorly understood. This study investigated the roles of oviductal estradiol-17β (E2) and progesterone (P4) concentrations on bovine oviductal functions in the luteal formation phase using RT-qPCR for some genes of oviductal epithelial cells. Bovine oviducts ipsilateral to the corpus luteum (CL) in the luteal formation phase were collected from a slaughterhouse. The concentration of oviductal E2 was positively correlated with the mRNA expressions of nuclear P4 receptor (PGR) and protein disulfide isomerase family A member 4 (PDIA4), which is related to protein secretion, in the ampulla and with estrogen receptor α (ESR1) mRNA expression in the isthmus. In contrast, the concentration of oviductal P4 was not correlated with oviductal mRNA expressions in either regions. Furthermore, for the candidate factor related to the oviductal E2 concentration, the CL parameters (CL size and tissue P4 concentration), first-wave dominant follicle (W1DF) parameters (follicle size and intrafollicular E2 concentration), and W1DF location (ipsilateral or contralateral to CL) did not influence the oviductal E2 concentration. In conclusion, our results suggest that the local oviductal E2 is a potential oviductal function regulator during the luteal formation phase.

Expression of H-ras, RLIP76 mRNA and Protein, and Angiogenic Receptors in Corpus Luteum Tissues during Estrous Cycles

Expression of H-ras, RLIP76 mRNA and Protein, and Angiogenic Receptors in Corpus Luteum Tissues during Estrous Cycles

VEGFR2 Expression Is Differently Modulated by Parity and Nulliparity in Mouse Ovary.

Parity and nulliparity exert opposite effects on women's health, as parity is considered a protective factor for several reproductive diseases. This study is aimed at determining if ovarian VEGF and VEGFR2 expression are differently modulated in the ovaries of parous and nulliparous mice. To this end primiparous and nulliparous fertile mice were sacrificed at postovulatory stage. Whole ovaries, corpus luteum, and residual stromal tissues were analyzed to assess VEGF/VEGFR2 expression levels. Ovarian mRNA amounts of Vegfa (120 and 164) and Vegfr2 were comparable between primiparous and nulliparous mice; both isoforms and receptor were accumulated mainly in corpus luteum tissues. VEGF 120 and 164 protein accumulation and distribution mirrored that of mRNA. Conversely, VEGFR2 protein content was significantly higher in ovaries of nulliparous mice and was more efficiently phosphorylated in ovaries of primiparous mice. In both groups, VEGFR2 was preferentially expressed in corpus luteum, while its phosphorylated form was equally distributed in two somatic compartments. We suggest that parity influences VEGFR2/phospho-VEGFR2 expression and tissue distribution. This difference could be part of a more complex mechanism that at least in mice is activated after the first pregnancy and likely aims to preserve female health.

Age-related changes in the bovine corpus luteum function and progesterone secretion.

Decreased fertility associated with maternal ageing is a well-known critical problem, and progesterone (P4) concentration decreases during the menopause transition in women. The corpus luteum (CL) secretes P4, thereby supporting the implantation and maintenance of pregnancy. It is proposed that a bovine model is suitable for studying age-associated decline of fertility in women because the physiology of cows is similar to that of women and cows have a greater longevity compared with other animal models. Thus, we investigated the age-dependent qualitative changes and inflammatory responses in the bovine CL. In vivo experiment: Cows were divided into three groups, namely, young (mean age: 34.8months), middle (80.1months) and aged (188.9months). Blood samples were collected on days 7 and 12 during the estrous cycle. In vitro experiments: Cows were divided into young (mean age: 27.6months) and aged (183.1months). The CL tissues of these groups were collected from a local slaughterhouse and used for tissue culture experiments. An in vivo experiment, plasma P4 concentration in aged cows was significantly lower than that in young cows, whereas no difference was found regarding the area of CL. An in vitro examination in the bovine CL tissues showed that the luteal P4 concentration, P4 secretion, and mRNA expression of StAR and 3β-HSD were lower in aged cows compared with young cows, especially in the early luteal phase. However, no differences were detected in the mRNA expression of inflammation- and senescence-related factors and inflammatory responses to lipopolysaccharides between the CL tissues from young and aged cows, indicating that an age-dependent increase in inflammation is not involved in the luteal function. P4 production and secretion from the bovine CL diminish in old cows, especially during the early luteal phase, suggesting that senescence may affect the luteal function in cows.

Peroxiredoxin I maintains luteal function by regulating unfolded protein response

BackgroundMounting evidence shows that ROS regulation by various antioxidants is essential for the expression of enzymes involved in steroidogenesis and maintenance of progesterone production by the corpus luteum (CL). However, the underlying mechanisms of peroxiredoxin 1 (PRDX1), an antioxidant enzyme, in luteal function for progesterone production in mice have not been reported. The aim of this study was to evaluate the functional link between PRDX1 and progesterone production in the CL of Prdx1 knockout (K/O) mice in the functional stage of CL.MethodsThe expression pattern of the unfolded protein response (UPR) signaling pathways, endoplasmic reticulum (ER) stress-induced apoptosis related genes and peroxiredoxins 1 (PRDX1) were investigated by western blotting analysis in CL tissue of 10 weeks mice during functional stage of CL. The protein levels of these genes after ER-stress inducer tunicamycin (Tm), ER-stress inhibitor tauroursodeoxycholic acid (TUDCA) and ROS scavenger, N-acetylcysteine (NAC) stimulation by intraperitoneal (i.p) injection were also investigated in CL tissue of wild type (WT) mice. Finally, we examined progesterone production and UPR signaling related gene expression in CL tissue of Prdx1 K/O mice.ResultsWe demonstrated that PRDX1 deficiency in the functional stage activates the UPR signaling pathways in response to ER stress-induced apoptosis. Interestingly, CL number, serum progesterone levels, and steroidogenic enzyme expression in Prdx1 K/O mice decreased significantly, compared to those in wild type mice. Levels of UPR signaling pathway markers (GRP78/BIP, P50ATF6, and phosphorylated (p)-eIF2) and ER-stress associated apoptotic factors (CHOP, p-JNK, and cleaved caspase-3) were dramatically increased in the CL tissue of Prdx1 K/O mice. In addition, administration of the NAC, reduced progesterone production and activated ER-stress-induced UPR signaling in the CL tissue obtained from the ovary of Prdx1 K/O mice. Taken together, these results indicated that reduction in serum progesterone levels and activation of ER-stress-induced UPR signaling are restored by NAC injection in the CL of Prdx1 K/O mice.ConclusionThese observations provide the first evidence regarding the basic mechanisms connecting PRDX1 and progesterone production in the functional stage of CL.

Changes in the expression of prostaglandin family members in bovine corpus luteum during the estrous cycle and pregnancy.

The aim of this study was to characterize certain prostaglandin family members in the bovine corpus luteum (CL) during the estrous cycle and pregnancy. The CL tissue was assigned to thestages 1-2, 3-4, 5-7, 8-12, 13-16 and >18 days (after regression) of the estrous cycle and1-2, 3-4, 6-7, and >8 monthsof pregnancy. In these samples, we investigated prostaglandin F2alpha (PTGF), prostaglandin E2 (PTGE), their receptors (PTGFR, PTGER2, and PTGER4), cyclooxygenase 2 (COX-2), PTGF synthase (PTGFS), and PTGE synthase (PTGES). The expression of messenger RNA (mRNA) was measured by reverse transcription quantitative polymerase chain reaction, hormones by enzyme immunoassay, and localization by immunohistochemistry. The mRNA expression of COX-2, PTGFS, and PTGES in CL during the early-luteal phase was high followed by a continuous and significant downregulation afterward, as well as during all phases of pregnancy. The concentration of PTGF in CL tissue was high during the early-luteal phase, decreased significantly in the mid-luteal phase, and increased again afterward. In contrast, the concentration of PTGE increased significantly during the late-luteal phase followed by a decrease during regression. The PTGE level increased again during late pregnancy. Immunohistochemically, the large granulose-luteal cells show strong staining for COX-2 and PTGES during the early-luteal stage followed by lower activity afterward. During pregnancy, most of the luteal cells were only weakly positive or negative. In conclusion, our results indicate that the examined prostaglandin family members are involved in the local mechanisms that regulate luteal function, specifically during CL formation, function, and regression and during pregnancy in the cow.

Angiocoupling between the dominant follicle and corpus luteum during waves 1 and 2 in Bos taurus heifers

The local two-way effect of the future and established dominant follicle (DF) and corpus luteum (CL) on color-Doppler signals of blood flow on each other (angiocoupling) was studied from ovulation to 16 days after ovulation during wave 1 and for the first time in wave 2 in Bos taurus heifers (N = 24). Identity of follicles greater than 4 mm was maintained from day to day. Blood-flow signals in the follicle wall and in the CL were estimated after scanning the entire structure. Ovarian patterns were defined based on the presence of a DF and CL: DF−CL pattern (DF and CL in the same ovary), DF pattern or CL pattern (DF and CL in opposite ovaries), and devoid pattern (neither DF nor CL). Comparisons were made of blood flow in each structure when in the same ovary vs opposite ovaries. Data were normalized to expected diameter deviation (DF closest to 8.5 mm). Blood-flow signals were detected in most growing follicles when they attained 6 mm (6.0–6.9 mm). Combined for waves 1 and 2, the percentage of all 6-mm growing follicles that displayed blood-flow signals was greater (P < 0.0001) for the DF−CL pattern (21/26, 81%) than for the patterns of DF alone, CL alone, or neither DF nor CL (total for the three patterns (17/42, 40.5%); there was no effect of left vs right ovaries. For each wave, the percentage of DF wall and percentage of CL tissue with blood-flow signals were greater for the DF−CL pattern vs the DF pattern or CL pattern. Percentage of DF wall with blood-flow signals for the DF−CL vs DF patterns was 30.0 ± 1.5% vs 19.5 ± 1.1% (P < 0.02) in wave 1 and 30.8 ± 1.3% vs 20.2 ± 1.1% (P < 0.0001) in wave 2. Percentage of CL tissue with blood-flow signals for the DF−CL vs CL patterns was 61.7 ± 2.4% vs 56.9 ± 1.7% (P < 0.03) in wave 1 and 78.8 ± 1.3% vs 74.0 ± 0.9% (P < 0.03) in wave 2. During wave 1, the percentage of CL tissue with blood-flow signals was greater (P < 0.002) when the DF and CL were in close proximity (distance between structures, < 2 mm; 66.9 ± 3.0%) than when separated (≥2 mm; 53.8 ± 3.5%). Normalization to the maximal DF diameter in wave 1 resulted in the novel finding that the percentage of blood-flow signals in the DF wall began to decrease 1 day before maximal diameter. Results supported the hypotheses that (1) percentage of DF wall and CL tissue with blood-flow signals in wave 2 is greater in the DF−CL pattern than in the DF or CL patterns and (2) percentage of DF wall with blood-flow signals in wave 1 decreases before the beginning of a decrease in diameter.

The expression of progesterone receptor coregulators mRNA and protein in corpus luteum and endometrium of cows during the estrous cycle

The aim of this study was to examine whether changes in the mRNA and protein expression of the progesterone receptor (PGR) coactivator P300/CBP-associated factor (PCAF) and the corepressor Nuclear Receptor Corepressor 1 (NCOR1) may participate in the regulation of PGR function during the estrous cycle in corpus luteum (CL) and endometrium and thus modulate the effect of progesterone (P4) within the reproductive system. The experimental material included CL and endometrial tissues from cows on days 2–5, 6–10, 11–16, and 17–20 of the estrous cycle. The mRNA expression of PCAF and NCOR1 was determined by means of real-time PCR, and protein levels were determined using western blotting. The highest mRNA and protein expression for PCAF (P<0.01) and NCOR1 (P<0.01) was found on days 6–16 in CL, whereas mRNA and protein expression for PCAF in endometrium was the highest on days 2–10 (P<0.05), but for NCOR1 it was the highest on days 2–5 (P<0.05) and decreased thereafter. Significant correlations were found between PCAF and NCOR1 mRNA and protein in CL and endometrium, between PCAF mRNA or protein and P4 levels only in CL, and between NCOR1 protein and P4 levels in endometrium only. Correlations between PCAF and NCOR1 mRNA and PCAF and NCOR1 protein were also found. These data suggest that the variable expression of these coregulators in CL and endometrium during the estrous cycle may depend on the influence of P4, and in these tissues both coregulators may compete for binding to the PGR.

Programmed necrosis - a new mechanism of steroidogenic luteal cell death and elimination during luteolysis in cows

Programmed necrosis (necroptosis) is an alternative form of programmed cell death that is regulated by receptor-interacting protein kinase (RIPK) 1 and 3-dependent, but is a caspase (CASP)-independent pathway. In the present study, to determine if necroptosis participates in bovine structural luteolysis, we investigated RIPK1 and RIPK3 expression throughout the estrous cycle, during prostaglandin F2α (PGF)-induced luteolysis in the bovine corpus luteum (CL), and in cultured luteal steroidogenic cells (LSCs) after treatment with selected luteolytic factors. In addition, effects of a RIPK1 inhibitor (necrostatin-1, Nec-1; 50 μM) on cell viability, progesterone secretion, apoptosis related factors and RIPKs expression, were evaluated. Expression of RIPK1 and RIPK3 increased in the CL tissue during both spontaneous and PGF-induced luteolysis (P < 0.05). In cultured LSCs, tumor necrosis factor α (TNF; 2.3 nM) in combination with interferon γ (IFNG; 2.5 nM) up-regulated RIPK1 mRNA and protein expression (P < 0.05). TNF + IFNG also up-regulated RIPK3 mRNA expression (P < 0.05), but not RIPK3 protein. Although Nec-1 prevented TNF + IFNG-induced cell death (P < 0.05), it did not affect CASP3 and CASP8 expression. Nec-1 decreased both RIPK1 and RIPK3 protein expression (P < 0.05). These findings suggest that RIPKs-dependent necroptosis is a potent mechanism responsible for bovine structural luteolysis induced by pro-inflammatory cytokines.

Estrogen metabolites in human corpus luteum physiology: differential effects on angiogenic activity

To determine tissue concentrations of E2, estrone, P, and estrogens metabolites (EMs) 2-methoxyestradiol, 2-methoxyestrone, 4-hydroxyestrone, and 16-ketoestradiol in corpus luteum (CL) of different ages, and after hCG administration; and to examine the effects of EMs on vascular endothelial growth factor (VEGF) secretion and angiogenic activity released by cultured luteinizing granulosa cells in the presence and absence of hCG. Experimental study. University. Thirty-two healthy women of reproductive age. Corpus luteum was collected at the time of minilaparotomy for tubal sterilization, at varying stages of the luteal phase (LP). Late-LP CL was collected 24hours after IM administration of 10,000 IU hCG. Granulosa cells were isolated from follicular aspirates obtained from healthy women participating in our IVF program for male factor infertility. Estrogen metabolite concentrations were determined in CL tissue, and VEGF was assessed in conditioned medium. The angiogenic activity was analyzed by bioassay. Concentrations of EMs with proangiogenic activity (16-ketoestradiol and 4-hydroxyestrone) were higher in early and mid-LP CL vs. late-LP CL. These EMs and hCG increased VEGF production and angiogenic activity. Conversely, late-LP CL had significantly higher levels of 2-methoxyestrone and 2-methoxyestradiol, which have antiangiogenic activity. Administration of hCG reduced the production of these EMs. Our findings suggest that the EMs are important paracrine modulators of CL function. Administration of hCG increases the production of EMs with proangiogenic activity and reduces the secretion of those EMs with antiangiogenic action, suggesting a novel mechanism by which the late-LP CL is rescued in conception cycles.

Type-dependent differences in Fas expression and phagocytes distribution in rat corpora lutea during natural regression: an immunohistochemical evidence.

Though Fas/Fas ligand (FasL) system-dependent apoptosis is considered to be the primary form of cell death in regressing corpus luteum (CL), the cellular identity and regulation of expression of the ligand and receptor molecules are not fully understood. Here, we focused on immunohistochemical determination of Fas expression during natural regression with comparison of three different types of rat CLs. Detected Fas was in good spatial association with cleaved caspase-3 and FasL proteins and with macrophages and neutrophils. In CLs of the cycle and pseudopregnancy, Fas-positive cell types included large and small luteal (steroidogenic) cells and capillary endothelial cells mainly, and blood-derived immune cells occasionally. Fas signals were abundant at multiple focal inflammatory-like sites. In contrast, Fas signals in CL of pregnancy did not localize in steroidogenic cells, but almost exclusively in endothelial cells and granulocytes. The signals scattered evenly throughout the CL tissue as phagocytes also did. In all CLs types, the numbers of Fas-expressing cells increased transiently after functional inactivation and at the early phase of structural regression. This observation revealed spatio-temporally regulated expression of Fas that was highly associated with apoptotic and phagocytotic systems and type-dependent differences in Fas expression and phagocytes dynamics in naturally regressing CL of rats.

Characterization of EST Gene in the Bovine Corpus Luteum during the Estrous Cycle.

The objective of this study was to investigate the expression of bovine luteum expressed sequence tags (ESTs), vascular endothelial growth factor (VEGF), and tumor necrosis factor receptor 1 (TNFR1) and the presence of functional ESTs in the bovine corpus luteum (CL) during different stages of the estrus cycle. Reverse transcription-polymerase chain reaction (RT-PCR) analysis showed a difference in the expression of ESTs during the CL stage. Concentration of ESTs in the CL tissue increased significantly from the mid-luteal stage and decreased thereafter. RT-PCR analysis showed higher levels of the EST genes in the CL of the mid-luteal stage than in other stages, and the same level of expression of VEGF. Immunohistochemistry analysis of the tissue from CL formation to regression showed low cytosol and aggregation of the nucleus. And activity caspase 3 (apoptosis detector) was most strongly detected in the CL1 stage of bovine. During the estrous cycle, the cytosol was magnified and differentiation of the nucleus was clearly manifested. The ESTs affected the CL, and the relationship between VEGF and TNFR1 played a pivotal role for CL development and activation, dependent on the stage of CL. These results suggest local production of ESTs, the presence of functional ESTs in the bovine CL, and that ESTs play a role in regulating the function of cell death in bovine CL.

Apoptosis-Related Factors in the Luteal Phase of the Domestic Cat and Their Involvement in the Persistence of Corpora Lutea in Lynx.

The corpus luteum (CL) is a transient gland formed in the ovary after ovulation and is the major source of progesterone. In the Iberian and Eurasian lynx, CL physiologically persist after parturition and retain their capacity to produce progesterone, thus suppressing the ovarian activity. This unique reproductive characteristic has a big impact on the success of assisted reproduction techniques in the endangered Iberian lynx. The mechanisms behind CL persistence are not yet understood and require extensive studies on potential luteotropic and luteolytic factors in felids. Because the apoptosis system has been shown to be involved in structural regression of CL in many species, we aimed to investigate the capacity of perCL to undergo apoptosis. In addition, we performed initial studies on the apoptosis system in the luteal phase of the domestic cat. No previous research on this system has been made in this species. Our factors of interest included agents of the intrinsic apoptosis pathway, i.e., pro-survival B-cell CLL/lymphoma 2 (BCL2) and pro-apoptotic BCL2-associated X protein (BAX), the executioner caspase-3 (CASP3), as well as of the extrinsic pathway, i.e., pro-apoptotic receptor FAS, and tumor necrosis factor (TNF) and its receptors (pro-apoptotic TNFRSF1A and pro-survival TNFRSF1B). We analyzed the relative mRNA levels of these factors, as well as protein localization of CASP3 and TNF during stages of pregnancy and the non-pregnant luteal phase in CL of domestic cats. The same factors were investigated in freshly ovulated CL (frCL) and perCL of Iberian and Eurasian lynx, which were histologically analyzed. All factors were present in the CL tissue of both domestic cat and lynx throughout all analyzed stages. The presence of pro-apoptotic factors BAX, CASP3, FAS and TNFRSF1A in perCL of the Eurasian and Iberian lynx might indicate the potential sensitivity of perCL to apoptotic signals. The expression of pro-survival factors BCL2 and TNFRSF1B was significantly higher in perCL compared to frCL of studied Iberian lynx, suggesting the potential involvement of these factors in the structural integrity of perCL. In both Iberian lynx and pregnant and non-pregnant domestic cats, the expression of TNFRSF1A was significantly higher in forming CL compared to other stages, suggesting the conserved involvement of this factor in the tissue reorganization during formation of the feline CL. The mRNA levels of CASP3 and TNFRSF1B were highest during regression stages of domestic cat CL. The current study provides initial results on the possible involvement of the apoptosis system in the structure and function of the feline CL and in its physiological persistence.

The luteotrophic function of galectin-1 by binding to the glycans on vascular endothelial growth factor receptor-2 in bovine luteal cells.

The corpus luteum (CL) is a temporary endocrine gland producing a large amount of progesterone, which is essential for the establishment and maintenance of pregnancy. Galectin-1 is a β-galactose-binding protein that can modify functions of membrane glycoproteins and is expressed in the CL of mice and women. However, the physiological role of galectin-1 in the CL is unclear. In the present study, we investigated the expression and localization of galectin-1 in the bovine CL and the effect of galectin-1 on cultured luteal steroidogenic cells (LSCs) with special reference to its binding to the glycans on vascular endothelial growth factor receptor-2 (VEGFR-2). Galectin-1 protein was highly expressed at the mid and late luteal stages in the membrane fraction of bovine CL tissue and was localized to the surface of LSCs in a carbohydrate-dependent manner. Galectin-1 increased the viability in cultured LSCs. However, the viability of LSCs was decreased by addition of β-lactose, a competitive carbohydrate inhibitor of galectin-1 binding activity. VEGFR-2 protein, like galectin-1, is also highly expressed in the mid CL, and it was modified by multi-antennary glycans, which can be recognized by galectin-1. An overlay assay using biotinylated galectin-1 revealed that galectin-1 directly binds to asparagine-linked glycans (N-glycans) on VEGFR-2. Enhancement of LSC viability by galectin-1 was suppressed by a selective inhibitor of VEGFR-2. The overall findings suggest that galectin-1 plays a role as a survival factor in the bovine CL, possibly by binding to N-glycans on VEGFR-2.

Ultrasound monitoring of blood flow and echotexture of the corpus luteum and uterus during early pregnancy of beef heifers

The aim was to characterize changes in the ultrasound characteristics of the CL and uterus in pregnant, inseminated nonpregnant, and cyclic beef heifers and to correlate findings with systemic progesterone (P4) concentrations with the intention of identifying possible markers for early identification of pregnancy. Heifers were randomly selected for artificial insemination after estrus synchronization. Ultrasound examinations of the CL and uterus were carried out by transrectal ultrasonography using a high-resolution ultrasound scanner equipped with a 12 MHz linear array probe on Days 7, 11, 14, 16, and 18 after artificial insemination (Day 0; i.e., estrus). Cross-sectional B-mode images of the CL were captured for calculation of CL tissue area and echotexture. Images of the CL and associated blood flow were captured and stored for analysis of luteal blood flow area and ratio. Longitudinal B-mode images of the uterine horns were captured just beyond the bifurcation of the uterine horns and stored for analysis of contrast and homogeneity (MaZda v4.6; Technical University of Lodz, Institute of Electronics, Poland). A total of three images were captured for each structure of interest. Serum concentrations of P4 were determined from blood samples collected at each ultrasound examination. After pregnancy diagnosis by ultrasound, heifers were retrospectively allocated as being pregnant (embryonic heartbeat on Day 28; n = 14) or nonpregnant (interestrous interval 18–21 days; n = 8) and their data were compared with noninseminated cyclic heifers (n = 10). Corpus luteum tissue area did not appear to change between pregnant, nonpregnant, or cyclic control groups between Days 7 and 18 (P > 0.05). No significant differences in CL echotexture characteristics were found between groups at any time point. There were no significant differences between pregnant, nonpregnant, and cyclic control groups for CL blood flow area (P > 0.05). However, CL blood flow ratio decreased significantly (P < 0.05) in both inseminated nonpregnant and cyclic heifers between Days 14 and 18, whereas it remained unchanged in pregnant heifers (P > 0.05). Uterine homogeneity was not significantly different between groups at any time point (P > 0.05). However, uterine contrast was significantly greater (P < 0.05) in pregnant compared with cyclic control heifers on Days 16 and 18. Concentrations of P4 were lower (P < 0.05) in nonpregnant and control heifers than in pregnant heifers from Days 16 to 18. In conclusion, there were differences between nonpregnant and cyclic heifers compared with pregnant heifers in P4 concentrations from Day 16. On Day 18, the CL and uterine characteristics were different between the nonpregnant and pregnant heifers. Ultrasound measures of CL blood flow and uterine echotexture may be useful to establish pregnancy status. Further investigation is required to identify if pregnancy diagnosis can be made on Day 18 or at a later day postpartum.

Profiling of luteal transcriptome during prostaglandin F2-alpha treatment in buffalo cows: analysis of signaling pathways associated with luteolysis.

In several species including the buffalo cow, prostaglandin (PG) F2α is the key molecule responsible for regression of corpus luteum (CL). Experiments were carried out to characterize gene expression changes in the CL tissue at various time points after administration of luteolytic dose of PGF2α in buffalo cows. Circulating progesterone levels decreased within 1 h of PGF2α treatment and evidence of apoptosis was demonstrable at 18 h post treatment. Microarray analysis indicated expression changes in several of immediate early genes and transcription factors within 3 h of treatment. Also, changes in expression of genes associated with cell to cell signaling, cytokine signaling, steroidogenesis, PG synthesis and apoptosis were observed. Analysis of various components of LH/CGR signaling in CL tissues indicated decreased LH/CGR protein expression, pCREB levels and PKA activity post PGF2α treatment. The novel finding of this study is the down regulation of CYP19A1 gene expression accompanied by decrease in expression of E2 receptors and circulating and intra luteal E2 post PGF2α treatment. Mining of microarray data revealed several differentially expressed E2 responsive genes. Since CYP19A1 gene expression is low in the bovine CL, mining of microarray data of PGF2α-treated macaques, the species with high luteal CYP19A1 expression, showed good correlation between differentially expressed E2 responsive genes between both the species. Taken together, the results of this study suggest that PGF2α interferes with luteotrophic signaling, impairs intra-luteal E2 levels and regulates various signaling pathways before the effects on structural luteolysis are manifest.