Luteal Cells Of Corpus Luteum Research Articles

Drp1 Expression and Phosphorylation in Steroidogenic Corpus Luteum during the Estrous Cycle in Rat Ovaries.

In response to luteinizing hormone (LH), a higher concentration of progesterone (P4) is produced in luteal cells of corpus luteum (CL). Mitochondria are an essential cellular organelle in steroidogenesis. The specific engagement of the concept regarding mitochondrial shaping with early stages of steroidogenesis was suggested in reproductive endocrine cells. Although the specific involvement of GTPase dynamin-related protein 1 (Drp1) with steroidogenesis has been demonstrated in luteal cells of bovine CL in vitro, its actual relationship with ovarian steroidogenesis during the estrous cycle remains unknown. In this study, while Fis1 and Opa1 protein levels did not show significant changes during the estrous cycle, Drp1, Mfn1, and Mfn2 proteins exhibited relatively lower levels at proestrus than at estrus or diestrus. 3β-HSD showed higher levels at proestrus than at estrus or diestrus. In addition, Drp1 phosphorylation (s637) was higher in proestrus than in estrus or diestrus. Immune-positive cells for Drp1, pDrp1 (s637), and 3β-HSD were all localized in the cytoplasm of luteal cells in the CL. The immune-positive cells for 3β-HSD were more frequently seen in the CL at proestrus than at estrus or diestrus. Immunoreactivity for Drp1 in luteal cells at proestrus was weaker than that at estrus or diestrus. However, pDrp1 (s637) immune-positive cells were mostly detected in luteal cells at proestrus. These results imply that steroidogenesis (P4 production) in the CL is closely related to phosphorylation of Drp1 at serine 637. Taken together, this study presents evidence that Drp1 phosphorylation at serine 637 is an important step in steroidogenesis in the CL.

Histological and micrometrical exploration of corpus Luteum in cyclic Nellore sheep

Ovaries from twenty two apparently healthy, non-pregnant and cyclic Nellore sheep was taken with the aim to explore histological and micrometrical aspects of the corpus luteum in ovary of adult Nellore sheep. The sections were cut at 5-6µ thick and histological procedures were carried out. From capsule, various trabeculae descend into the substance of corpus luteum. Thickness of the trabeculae which descends from the capsule was almost equal in thickness to that of capsule at the periphery and tapers towards the centre. The capsule mainly comprised of collagen and reticular fibres. The occurrence of two fully developed corpora lutea in the same ovary was also observed. On the basis of shape and the size, three basic types of lutein cells were observed: large (16.38-28.17µm), small (7.95-27.06µm) and spindle shaped (15.73-29.57µm) cells. Moderate PAS reaction was seen in Capsule, Trabeculae and mild reaction was seen in luteal cells of corpus luteum.

Expression and localization of cellular FLICE-like inhibitory protein (cFLIP), an anti-apoptotic factor, in corpora lutea during the estrous cycle and pregnancy in Thai swamp buffalo (Bubalus bubalis).

In female mammals, luteal cells rapidly proliferate and form corpora lutea (CLs) after ovulation. The corpus luteum (CL) plays crucial roles in establishing and maintaining pregnancy. To gain further insights into the role of cellular FLICE-like inhibitory protein (cFLIP), an anti-apoptosis factor that is structurally similar to procaspase-8 but lacks proteolytic enzyme activity, we examined the expression in CLs of Thai swamp buffalos (Bubalus bubalis) during the early, mid, and late stage of the estrous cycle and pregnancy. cFLIP short form and long form (cFLIPS and cFLIPL, respectively) mRNA and protein levels were assessed by reverse transcription-polymerase chain reaction and western blotting, respectively. cFLIPS mRNA levels were low in the mid and late stages of the estrous cycle and increased during pregnancy (P < 0.05). cFLIPL mRNA was highly expressed in CLs during pregnancy and was lower in the mid and late stages of the estrous cycle. The level of cFLIPS protein was high in CLs during pregnancy and low levels were noted in the mid stage of the estrous cycle (P < 0.05). Higher levels of cFLIPL protein were demonstrated in CLs during pregnancy and lower levels were found in CLs during the early stage of the estrous cycle. Strong positive immunohistochemical staining for cFLIPS/L proteins was observed in luteal cells during pregnancy. The present findings revealed that cFLIP was at the highest level in CLs during pregnancy, and this may act as a dominant survival anti-apoptotic factor by inhibiting intracellular apoptosis signal transduction in luteal cells of CLs during pregnancy.

Timely expression and activation of YAP1 in granulosa cells is essential for ovarian follicle development.

Although the role of the Hippo signaling pathway in development and tumorigenesis has been extensively studied in multiple organs, its role in ovarian follicle development remains largely unknown. Here, we report that Yes-Associated Protein 1 (YAP1), the major effector of Hippo signaling, is spatiotemporally expressed in ovarian granulosa cells and plays a critical role in the regulation of follicle development. We found that the active form of YAP1 (nuclear YAP1) was predominantly expressed in proliferative granulosa cells, whereas the inactive form of YAP1 (cytoplasmic YAP1) was mainly detected in luteal cells (terminally differentiated granulosa cells). Pharmacological inhibition of YAP1 activity disrupted mouse ovarian follicle development in vitro and in vivo. Foxl2 promoter-driven knockout of Yap1 in ovarian granulosa cells resulted in increased apoptosis of granulosa cells, decreased number of corpora lutea, reduced ovarian size, and subfertility in transgenic mice. However, Cyp19a1 promoter-driven knockout of Yap1 in differentiated granulosa cells of preovulatory follicles and luteal cells of corpora lutea had no effect on ovarian morphology and fertility. Mechanistic studies demonstrated that YAP1 interacted with epidermal growth factor receptor and TGF-β signaling pathways to regulate granulosa cell proliferation, differentiation, and survival. Results from this study identify YAP1 as a critical regulator of granulosa cell proliferation and differentiation. Balanced expression and activation of YAP1 is essential for follicle development and successful reproduction. YAP1 is a promising target for treatment of subfertility associated with abnormal granulosa cell function.-Lv, X., He, C., Huang, C., Wang, H., Hua, G., Wang, Z., Zhou, J., Chen, X., Ma, B., Timm, B. K., Maclin, V., Dong, J., Rueda, B. R., Davis, J. S., Wang, C. Timely expression and activation of YAP1 in granulosa cells is essential for ovarian follicle development.

The Effects of Unpredictable Stress on the LHR Expression and Reproductive Functions in Mouse Models

Sung-Young Choi, Jin-Heum Park, Yuxia Zhu, Young-Jong kim, Jae-Ok Park, Changjong Moon, Taekyun Shin, Meejung Ahn, Suk-Soo Kim, Young-Sik Park, Hyung-Bok Chae, Tae-Kyun Kim and Seung-Joon Kim. J Vet Clin 2014;31:394-402. https://doi.org/10.17555/ksvc.2014.10.31.5.394

Direct actions of ACTH on ovarian function of pseudopregnant rabbits

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

Higher expression of hyaluronan binding protein 1 (HABP1/p32/gC1qR/SF2) during follicular development and cumulus oocyte complex maturation in rat

Ovulation is a complex process of releasing a fertilizable oocyte and depends on the proper formation of an extracellular hyaluronan rich matrix by the cumulus oocyte complex (COC). The formation of a HA rich matrix is dependent on the synthesis and organization of HA in the presence of several biomolecules that mediate its crosslinking. To gain an insight into the follicular maturation and COC expansion, we have studied the expression of hyaluronan binding protein 1 (HABP1), which is known to interact specifically with hyaluronan. The level of HABP1 increased markedly during ovulation after gonadotropin stimulation, and the overexpression was seen in mural granulosa cells, expanding cumulus cells and follicular fluid. However, HABP1 could not be detected in the luteal cells of corpus luteum after ovulation. Such increased expression of HABP1 was observed both during in vivo and in vitro conditions of COC expansion. The level of HABP1 transcript was upregulated up to fivefold after COC expansion as compared to compact COC. Immunofluorescence analysis showed HABP1 to be localized in the cytoplasm and extracellular matrix, suggesting its role in ECM organization. The cultured expanded COC treated with hyaluronidase for different time periods showed the gradual dispersion of COC, which coincide with the loss of HABP1 from the matrix suggesting that HABP1 is bound to hyaluronan. These results indicate that HABP1 expressed in rat COCs during maturation may facilitate the formation of the HA matrix in the extracellular space around the oocyte with cumulus expansion during maturation.

Melanoma cell adhesion molecule (MCAM/CD146) is expressed on human luteinizing granulosa cells: enhancement of its expression by hCG, interleukin-1 and tumour necrosis factor-alpha.

Melanoma cell adhesion molecule (MCAM) was originally reported to be involved in the invasion and progression of melanoma. It was also shown to be responsible for the attachment of cells to endothelial cells. In this study, we demonstrated by immunohistochemistry that immunoreactive MCAM was not expressed on granulosa cells in the pre-ovulatory follicle, but it was clearly detected in large luteal cells in corpora lutea from the mid-luteal phase of the menstrual cycle. Northern blotting analysis confirmed the expression of MCAM mRNA in corpus luteum. MCAM was weakly detected by immunocytochemical staining in human luteinizing granulosa cells isolated from patients undergoing IVF treatment. Its expression was found to be increased during time in culture of these cells. Flow cytometry and Northern blot analysis revealed that MCAM expression on luteinizing granulosa cells was enhanced when the cells were cultured for 5 days in the presence of hCG (1 IU/ml) or cytokines such as interleukin-1alpha (10 ng/ml) and tumour necrosis factor-alpha (10 ng/ml). No significant difference of MCAM expression was observed between the cultures under normoxic (20% oxygen) and hypoxic (1% oxygen) conditions. These results indicate that luteinizing granulosa cells express MCAM and that MCAM expression is regulated by LH/hCG and cytokines during luteinization. Since MCAM has been reported to mediate cellular interaction with endothelial cells, this molecule may play a role in neovascularization during corpus luteum formation in the human ovary.

A differentiation antigen of human large luteal cells in corpora lutea of the menstrual cycle and early pregnancy.

To investigate the mechanism(s) of luteal cell differentiation, we raised a monoclonal antibody, HCL-1, against human large luteal cells. The antigen was undetectable in growing and preovulatory follicles by immunohistochemistry. The antigen was initially detected on centrally located luteinizing granulosa cells during CL formation. During the midluteal phase, the antigen was expressed at high levels on large luteal cells. Large luteal cells in the CL during late luteal phase and early pregnancy also expressed high levels of HCL-1 antigen, whereas small luteal cells at any stage of the CL did not. Granulosa cells in some atretic follicles weakly expressed HCL-1 antigen. Immunofluorescence staining of enzymatically dispersed luteal cells from human mature CL revealed that HCL-1 antigen was present on the cell surface of large luteal cells. Human granulosa cells isolated from patients who had undergone in vitro fertilization treatment were cultured for 7 days. Indirect immunofluorescence detected HCL-1 antigen on only a few granulosa cells after culture for 1 day and on almost all granulosa cells after culture for 7 days. Flow cytometry of 7-day-cultured cells showed that the percentages of positivity for HCL-1 antigen as well as the mean fluorescence intensities of granulosa cells cultured with hCG (1 IU/ml) were significantly lower than those of the controls (without treatment) (44.3 +/- 3.2% vs. 62.9 +/- 4.0%. p < 0.01; 60.9 +/- 6.7 vs. 82.1 +/- 7.6, p < 0.05). By contrast, the mean fluorescence intensities of cells cultured with interleukin- 1 alpha (10 ng/ml, 105 +/- 6.3, p < 0.05) and tumor necrosis factor alpha (10 ng/ml, 112 +/- 11.2, p < 0.05) were significantly higher than those of controls. These findings showed that the cell surface antigen detected on human large luteal cells by HCL-1 was differentiation-related, and that large luteal cells in the CL of pregnancy are derived from granulosa cells via large luteal cells in the CL of the menstrual cycle.

Changes in the gene expression of a protein with the cdc10/SWI6 motif, V-1, during rat follicular development and corpus luteum formation.

We examined the gene expression of V-1, a novel soluble protein with the cdc10/SWI6 motif, in pseudopregnant rat ovaries. Northern blot analysis on days 1, 5, and 11 of pseudopregnancy revealed an approximately 2-fold increase in the V-1 messenger RNA (mRNA) expression level on day 5 to that on day 1, and no significant change was observed between those on day 5 and day 11. An injection of human CG on days 5 further increased th V-1 mRNA level to about 1.6-fold of that of the untreated control. Western blot analysis showed higher V-1 protein expression on days 5 and 11 of pseudopregnancy than that on day 1. In situ hybridization and immunohistochemistry with ovaries on day 3 of pseudopregnancy showed that luteal cells of corpora lutea and also cells of the coexisting follicles including oocytes express V-1 mRNA and the protein, with apparent rank order of the expression: oocytes > luteal cells > follicular cells >> atretic follicular cells including oocytes. These data indicate the dynamic change in the V-1 gene expression in the ovarian steroidogenic cells and oocytes and suggest potential roles of the V-1 protein in ovarian functions including corpus luteum formation and folliculogenesis.

Immunolocalization of transforming growth factor-beta 1 and transforming growth factor-beta 2 in the mouse ovary during gonadotrophin-induced follicular maturation.

An immunohistochemical approach was utilized to evaluate the cellular distribution of transforming growth factor-beta 1 (TGF beta 1) and transforming growth factor beta 2 (TGF beta 2) at different stages of follicle development in the prepubertal mouse ovary under the following conditions: (i) after pregnant mare's serum gonadotrophin (PMSG) treatment; (ii) after PMSG and human chorionic gonadotrophin (HCG) treatment; (iii) after PMSG and HCG treatment plus mating. In the immature ovary, TGF beta 1 and TGF beta 2 immunoreactivities are localized in theca and granulosa cells and in oocytes. After PMSG treatment, TGF beta 1 and TGF beta 2 immunoreactivities are localized in granulosa cells; in addition, TGF beta 2 staining is noted in the matrix surrounding antral cells. Staining for both TGR beta 1 and TGF beta 2 drops in the theca but persists in the oocyte. PMSG plus HCG treatment results in a significant increase in TGF beta 1 and TGF beta 2 immunoreactivity in the theca and in the maintenance of TGF beta 1 staining in both basal granulosa cells and cumulus cells whereas TGF beta 2 immunoreactivity is essentially localized in the matrix surrounding cumulus cells. Staining for TGF beta 1 and TGF beta 2 persists in the oocyte. Following PMSG plus HCG treatment and mating, TGF beta 1 immunoreactivity is localized in the luteal cells of corpora lutea and TGF beta 2 shows a similar localization pattern. This study provides evidence that TGF beta 1 and TGF beta 2 peptides are expressed in specific cell types during induced follicular maturation in the mouse ovary.

Cloning and expression of a cDNA for mouse prostaglandin F receptor.

A functional cDNA clone for mouse prostaglandin (PG) F receptor was isolated from a mouse cDNA library using polymerase chain reaction based on the sequence of cloned prostanoid receptors, and cross-hybridization screening. The mouse PGF receptor consists of 366 amino acid residues with putative seven transmembrane domains. The sequence revealed the highest homology to the EP1 subtype of PGE receptor and thromboxane (TX) A2 receptor. Ligand binding studies using membranes of COS cells transfected with the cDNA revealed specific [3H]PGF2 alpha binding. The binding was displaced with unlabeled PGs in the order of PGF2 alpha = 9 alpha, 11 beta PGF2 > PGF 1 alpha > PGD2 > STA2 (a stable TXA2 agonist) > PGE2 > iloprost (a stable PGI2 agonist). PGF2 alpha increased inositol trisphosphate formation in a concentration-dependent manner in COS cells expressing PGF receptor. RNA blot and in situ hybridization analyses demonstrated that the PGF receptor transcripts are abundantly expressed in luteal cells of corpus luteum and in a lesser amount in kidney, heart, stomach, and lung.

Human luteal cells express dipeptidyl peptidase IV on the cell surface.

We previously reported that human theca interna cells and small luteal cells express membrane-bound aminopeptidase N, and suggested that membrane-bound peptidases are involved in folliculogenesis and luteal function by regulating extracellular peptide concentrations. In this study, we examined the expression of dipeptidyl peptidase IV (DPP IV), which is a membrane-bound peptidase and has its catalytic domain at extracellular sites, in human granulosa cells, thecal cells of growing, preovulatory, and atretic follicles, as well as corpora lutea. Indirect immunofluorescence staining of ovarian tissues with specific monoclonal antibodies revealed that DPP IV was present in large and small luteal cells in corpora lutea. DPP IV peptidase activity was also detected histochemically in corpora lutea. In growing, preovulatory, and atretic follicles, there was weak immunoreactivity and DPP IV peptidase activity on luteinized theca interna cells, but not on granulosa cells. The expression of DPP IV on the cell surface of large and small luteal cells was confirmed by indirect immunofluorescence staining of freshly isolated luteal cells. These results indicate that DPP IV is a useful surface differentiation marker of human luteal cells and suggest that peptidases are involved in luteal function.

Distribution of epidermal growth factor receptors in the rat ovary

The distribution of epidermal growth tactor (EGF) was studied in the ovary using light microscope radioautography which was performed at different time intervals (2–60 min) after intravenous (i.v.) injection of [ 125I]EGF into adult rat at random stages of the estrous cycle and also after topical localization of iodinated EGF on slide-mounted frozen ovarian sections. After i.v. injection of label, the labeling was mostly observed in the theca interna cells of secondary, preovulatory and atretic follicles and luteal cells of corpus luteum. Primordial and primary follicles did not show any significant labeling. The time-course study performed on luteal cells showed that, 2 min after injection, most silver grains were found at the periphery of the cells. At the 10 min time interval, silver grains were found both at the periphery and over the cytoplasm of these cells. The number of grains was very reduced over the cytoplasm at the 60 min time interval. In the in vitro study, a positive radioautographic reaction was seen in the same cellular elements as found in vivo, with the additional labeling of the granulosa cells of growing and preovulatory follicles. Control experiments indicated that the radioautographic labeling was due to specific interaction of [ 125I]EGF with its receptor. These results clearly indicate that EGF binding sites are present in luteal, thecal and granulosa cells, and provide support for the inhibitory and stimulatory actions of EGF on different parameters of ovarian cells.

Monoclonal antibody recognition of estrogen receptor in rabbit corpus luteum

We demonstrated previously by administering [3H]estradiol and using autoradiography that, in vivo, the exogenous estradiol that will sustain progesterone production is sequestered to nuclei of the luteal cells in corpora lutea of pseudopregnant rabbits. Our objective in the present experiments was to use an immunological method to demonstrate that estrogen receptor of the rabbit corpus luteum is associated with this uptake. For this purpose, we prepared nuclear extracts from corpora lutea removed from anesthetized pseudopregnant rabbits 10 min after arterial infusion of 125I-estradiol. These extracts were then analyzed by sucrose density gradient centrifugation in the presence and absence of monoclonal antibody against human breast cancer estrogen receptor. We found that the 125I-estradiol-binding component extracted with 0.5 M KCl from the nuclear pellet sediments in the 3-4S region in high-salt gradients; in the presence of monoclonal antibody (D-547 Sp2 gamma), the entire estradiol-binding component is shifted to the 8-9S region, indicating the formation of immunocomplex with the 125I-estradiol-receptor moiety. The principal estradiol-binding component that can be observed in the 3-4S region of high-salt gradients of cytosol of rabbit corpora lutea reacts with this antibody regardless of whether 125I- or [3H]estradiol is given in vivo or in vitro. In both cytosol and nuclear extracts of luteal tissue, unlabeled diethylstibestrol totally inhibits the binding of radiolabeled estrogen to this antibody-shifted 125I- or [3H]estradiol-binding moiety; neither progesterone nor testosterone inhibit binding of radiolabeled estrogen to the antibody-shifted moiety. From these findings we conclude that sequestering of estradiol by the rabbit corpus luteum in vivo is accomplished by the steroid-specific estrogen receptor that shares characteristics of the estrogen receptor found in human breast cancers, including a) antigenic features and b) the capacity to translocate estrogens from the cytosol to the nuclear fraction.

Evidence for relaxin in corpora lutea of late pregnant cows.

Relaxin was isolated from corpora lutea of third trimester pregnant cows. Fractions having relaxin activity eluted in the 6000 and 1400 molecular weight ranges when chromatographed on Bio-Gel P-10. Both fractions were shown to inhibit spontaneous contractions of the mouse uterus, and the 6000 molecular weight fraction promoted formation of the mouse interpubic ligament. Agar diffusion studies with an antiserum to purified porcine relaxin showed a continuous precipitin line with no spurring between the two bovine relaxin fractions and NIHR-P1 porcine relaxin standard. Electrofocusing of the 6000 molecular weight fraction demonstrated three fractions which were capable of inhibiting contractions of the mouse uterus and which cross-reacted with porcine relaxin antiserum. The isoelectric points of these three fractions were 8.8, 10.1, and 11.5. Large luteal cells in corpora lutea from cows in the midtrimester of pregnancy were detected that gave a positive stain for relaxin with the immunoperoxidase method.

Ultrastructure of the human ovary: II. The luteal cell at term

The ultrastructure of luteal tissue obtained from term cesarean section deliveries is described. The cytoplasmic organelles of the term luteal cells appeared well preserved and intact. The agranular endoplasmic reticulum (ER) compared favorably with that reported in luteal cells of corpora lutea of the menstrual cycle. Localized concentrations of an atypical granular ER are described and its continuity with the agranular ER was recognized. Mitochondria and Golgi complex are well developed and without notable features. Large homogenous globules are frequently seen in luteal cell cytoplasm; these are assumed to represent the “colloid droplets” described by light microscopy. Lipid globules are rarely seen in term luteal cells and the implications of this are discussed. On the basis of the ultrastructure of the term luteal cells, it is concluded that they are not highly degenerate cells and they could be capable of secretion.