Morphological characteristics of the bovine corpus luteum during the estrous cycle and pregnancy

The corpus luteum, one of the biological clocks of the estrous cycle and pregnancy, is known foremost for its production of progesterone that blocks the pituitary release of gonadotropins and prepares the uterus for a pregnancy. The cellular sources of this progesterone are the steroidogenic small and large luteal cells. Other luteal cells that are not steroidogenic, but are believed to have an important role in the function of this gland are the fibroblast, macrophages and endothelial cells. The most prominent luteal cell is the large steroidogenic cell characterized by an abundance of smooth endoplasmic reticulum and densely packed spherical mitochondria that are indicative of its contribution to most of the circulating progesterone believed to be constitutively secreted and not under the control of LH. Other distinguishing features of the large luteal cell are the presence of rough endoplasmic reticulum, prominent Golgi, and secretory granules that are indicative of endocrine cells. This cell undergoes dynamic changes across the estrous cycle and pregnancy, believed to reflect a change in progesterone and protein secretion that will eventually influence a successful pregnancy or another ovulation if pregnancy fails. The morphological characteristics of the bovine luteal cells are the focus of this review.     

Differences of microvascular endothelium in the bovine corpus luterum of pregnancy and the corpus luteum of the estrous cycle

Summary— The purpose of the present study was to investigate potential modulations of endothelial cells of the bovine corpus luteum (CL) during pregnancy. Luteal endothelia of pregnant and non-pregnant cows were isolated and purity of cultures was verified by flow cytometric quantification of three independent endothelial markers (von Willebrand factor, angiotensin converting enzyme, Bandeiraea simplicifolia agglutinin I ligands). Different cellular parameters including light and electron microscopical investigation of morphology and growth characteristics as well as quantification of cellular lectin binding sites were compared. Extensive heterogeneity between luteal endothelial cells in pregnant and non-pregnant animals could be demonstrated, reflected in functional attributes like angiogenic activity, ultrastructural characteristics and the quantitative expression of cellular carbohydrates. Two different morphological types of cells (‘cobblestone growth pattern’ and ‘arcuate growth pattern’) were isolated from the CL of pregnancy as well as from the cyclic CL. Spontaneous angiogenic activities, including cellular migration in band-like structures and formation of ring-like structures, were observed in endothelial cells isolated from the CL of pregnant cows exclusively. This strongly suggests that microvascular luteal endothelium of pregnant animals, in contrast to the one of non-pregnant animals, is able to produce quantitatively and/or qualitatively specific angiogenesis factor(s). Heterogeneity between luteal endothelial cells in the pregnant and non-pregnant animal could also be demonstrated by quantification of lectin (Bandeiraea simplicifolia agglutinin I, concanavalin A, Dolichos biflorus agglutinin, Ulex europaeus agglutinin I, wheat germ agglutinin) binding sites: quantitative expression of specific endothelial cell surface carbohydrates could be correlated to be status of pregnancy, thus emphasizing the actual need of quantification of lectin binding.     

Lipoprotein lipase activity in the bovine corpus luteum during the estrous cycle and early pregnancy.

Lipolytic activity measured at pH 8.6 in bovine corpora lutea exhibited classical properties of lipoprotein lipase (LPL) in terms of serum and heparin stimulation and NaCl inhibition. LPL activity was measured in 23 corpora lutea collected at different stages of the estrous cycle and early pregnancy. The LPL activity in cyclic corpora lutea (mumole FA released/hr/100 mg acetone powder) was low at Days 4-8 of the estrous cycle (3.1 +/- 1.5: mean +/- SE) and at Days 19-20 (1.6 +/- 0.6). However, high activity of the enzyme was found at Days 12-15 of the cycle (11.8 +/- 1.8); these concentrations were significantly (P less than 0.01) elevated over those found at Days 4-8 and 19-20. The enzyme activity began to decline at Days 16-18 of the estrous cycle (5.1 +/- 1.7). Low enzyme activity was found in the corpora lutea removed from two cows at Day 22 of pregnancy. Progesterone concentrations were measured in 16 of the 23 corpora lutea and a good correlation (r = 0.75, P less than 0.01) was found between lipoprotein lipase and progesterone concentrations of the tissue. The data suggest that LPL may be involved in controlling the transfer of fatty acids, including arachidonic, from plasma lipoproteins to luteal tissue.     

Interrelationships among morphology, echotexture, and function of the bovine corpus luteum during the estrous cycle

It has been suggested that ultrasound image attributes are a potential indicator of physiological and functional status of the corpus luteum (CL) in several species, including cattle. The aims of this study were to evaluate CL morphological, functional and echotextural characteristics, and also to investigate the hypothesis that those attributes are correlated and change similarly throughout an estrous cycle. Ovaries of crossbred (Bos taurus taurus × Bos taurus indicus) heifers were evaluated using ultrasonography daily throughout an interestrus interval using a B-mode, real-time ultrasound machine equipped with a 5 MHz linear-array rectal transducer, during a natural estrous cycle (Experiment 1; n = 12) or during a shortened cycle, with luteolysis induction 10 d after estrus (Experiment 2; n = 6). Blood samples were collected for assay of plasma progesterone concentrations. Corpora lutea areas were measured and daily images of each CL were videotaped and digitized for computer-assisted analysis using custom-developed software. In Experiment 1, area of luteal tissue increased until a maximum value 10 d after estrus (P < 0.001), followed by a plateau phase, and then a decline beginning 14 d after estrus. Luteal tissue area was highly correlated to plasma progesterone concentrations (r = 0.86; P < 0.001). When luteolysis was induced in Experiment 2, loss of CL function (decrease in plasma progesterone concentrations to metestrous values) preceded tissue regression by 48 h (24 h compared with 72 h; P < 0.001). The mean pixel value of ultrasound images did not change in Experiment 1 (P > 0.70), but a day effect on this attribute was observed in Experiment 2 (P = 0.052). In contrast, mean pixel value was correlated to plasma progesterone concentrations in Experiment 1 (r = −0.63; P < 0.05), but not in Experiment 2 (r = −0.28; P > 0.10). In regard to CL heterogeneity, defined as the standard deviation of the mean pixel value of the luteal tissue, a time effect was observed following both natural (Experiment 1; P < 0.009) and luteolysis-induced (Experiment 2; P < 0.05) estrous cycles (P < 0.05). Moreover, this variable was correlated with plasma progesterone concentrations (r = −0.71 and −0.58 in Experiments 1 and 2, respectively; P < 0.01), indicating that CL images were more heterogeneous during metestrus and after luteolysis (functional regression). In summary, morphological and echotextural attributes were correlated with CL function and underwent similar changes during the estrous cycle. Luteal tissue heterogeneity, assessed by ultrasonography, is considered a potential indicator of CL functional status, because it is correlated to circulating progesterone concentrations.     

Expression pattern of prokineticin 1 and its receptors in bovine ovaries during the estrous cycle: involvement in corpus luteum regression and follicular atresia

Prokineticin 1 (PROK1), also termed endocrine gland-derived vascular endothelial growth factor (endocrine gland-derived VEGF), is a newly identified protein assigned with diverse biologic functions. It binds two homologous G protein-coupled receptors, PROKR1 and PROKR2. To better understand the roles of PROK1 and its receptors in ovarian function, their expression was determined in follicles and corpora lutea (CLs) at different developmental stages. PROK1 mRNA levels were low at early luteal stage and midluteal stage, but increased sharply during natural or induced luteolysis. High PROK1 mRNA levels also were found in atretic follicles. This profile of PROK1 expression was opposite to that of the well-established angiogenic factor VEGF. Of the two receptor-type expressions, PROKR1 but not PROKR2 was correlated positively with its ligand. Immunohistochemical staining revealed that PROK1 was located mainly within the muscular layer of arterioles, and during regression it also was localized to macrophages and steroidogenic cells. The expression pattern of ITGB2 mRNA, a leukocyte cell marker, overlapped that of PROK1, thus suggesting that leukocyte infiltration may explain the elevated expression of PROK1 in atretic follicles and regressing CL. Indeed, flow cytometry analyses showed that nearly all beta-2 integrin chain (ITGB2)-positive cells also were stained with anti-PROK1 and that significantly more ITGB2/PROK1 double-stained cells were present in degenerating follicles and CL. Furthermore, when challenged in vitro with PROK1, adherent, mononuclear cell numbers and TNF levels were elevated, indicating that PROK1 triggers monocyte activation. Together, these data suggest that PROK1, acting via PROKR1, may be involved in the recruitment of monocytes to regressing CL and atretic follicles and their consequent activation therein.     

Different expression of PGE synthase,PGF receptor,TNF, Fas and oxytocin in the bovine corpus luteum of the estrous cycle and pregnancy

Functional differences between the corpus luteum (CL) of pregnancy and CL of the cycle in cows were examined. Messenger RNA and protein levels of prostaglandin (PG) E synthase (PGES), PGF2α receptor (PGFR), tumor necrosis factor-α (TNF) and Fas were found to be higher in the CL of pregnancy than in CL of the cycle. Oxytocin (OT) mRNA and protein levels were lower in the CL of pregnancy. Messenger RNA levels of progesterone receptor (PR), luteinizing hormone receptor (LHR), PGE2 receptor (PGER), PGF synthase (PGFS), TNF receptor type I (TNFRI) and TNF receptor type II (TNFRII) did not differ between the cycle and pregnancy. PGE2 and PGF2α production by cultured bovine endometrial tissues was decreased by a supernatant derived from the homogenized CL of pregnancy but not by that of the CL of the cycle, suggesting that specific substances in the CL of pregnancy affect endometrial PG production in cows. Collectively, PGES, PGFR, TNF, Fas or OT may contribute to differences between the CL of pregnancy and CL of the estrous cycle in cows.     

Morphometric analysis of cell types in the ovine corpus luteum throughout the estrous cycle

The cellular composition of ovine corpora lutea obtained during the early (Day 4), mid (Days 8 and 12), and late (Day 16) stages of the estrous cycle was determined by morphometric analysis. Individual corpora lutea were collected via midventral laparotomy from a total of 19 ewes. A center slice from each corpus luteum was processed for electron microscopy and subsequent morphometric analysis of the numbers and sizes of steroidogenic and nonsteroidogenic cells. Luteal weight progressively increased throughout the estrous cycle (p less than 0.05). Corpora lutea collected on Day 16 were assigned to one of two subgroups on the basis of gross appearance and weight: nonregressed (NR, 542 +/- 25 mg) or regressed (R, 260 +/- 2 mg). There were no significant changes in the proportion of the corpus luteum occupied by small luteal cells (19 +/- 2%) or large luteal cells (36 +/- 1%) throughout the estrous cycle. The total number of steroidogenic cells per corpus luteum increased from 21.8 +/- 3.7 (X 10(6)) on Day 4 to 61.7 +/- 5.4 (X 10(6)) on Day 8 (p less than 0.05) and remained elevated thereafter. The number of small luteal cells was 10.0 +/- 2.7 (X 10(6)), 39.7 +/- 1.4 (X 10(6)), 46.1 +/- 5.8 (X 10(6)), 49.0 +/- 13.7 (X 10(6)), and 29.9 +/- 8.6 (X 10(6)) on Days 4, 8, 12, 16 (NR), and 16 (R), respectively (p less than 0.05, Day 4 vs. Days 8, 12, 16 NR). In contrast, the number of large luteal cells was 11.8 +/- 1.5 (X 10(6)) on Day 4 and did not vary significantly during the remainder of the estrous cycle. The numbers of nonsteroidogenic cell types increased (p less than 0.05) from Day 4 to Day 16 (NR) but were decreased in regressed corpora lutea (Day 16 R). Regression was characterized by a 50% decrease (p less than 0.05) in the total number of cells per corpus luteum from 243 +/- 57 ( X 10(6)) on Day 16 (NR) to 125 +/- 14 ( X 10(6)) on Day 16 (R) (p less than 0.05). Small luteal cells remained constant in volume throughout the entire estrous cycle (2520 +/- 270 microns 3), whereas large luteal cells increased in size from 5300 +/- 800 microns 3 on Day 4 to 16,900 +/- 3300 microns 3 on Day 16 (NR) (p less than 0.05). In summary, small luteal cells increased in number but not size throughout the estrous cycle, whereas large luteal cells increased in size but not number.     

Immunohistochemical localization of estrogen receptors in the ovine corpus luteum throughout the estrous cycle

Using immunohistochemistry and Western blot analysis we attempted to identify the estrogen receptors in ovine luteal cells at different stages of the estrous cycle. Monoclonal antibody against estrogen receptors was used for immunolocalization of estrogen receptor-alpha in corpora lutea sections. Generally, the most intense cytoplasm staining was present in large luteal cells. On the 6th day of the estrous cycle, weak immunostaining of estrogen receptors was observed in large luteal cells as well as in the connective tissue. Luteal cells from regressing corpora lutea expressed the weakest immunostaining. The most intense immunoreactivity for estrogen receptors was found in sections of corpora lutea collected on the 9th day of the cycle. Both, cytoplasmic and nuclear localization was observed depending on cell types in the ovine corpus luteum. Our studies demonstrated the presence of the estrogen receptor-alpha in the luteal cells and suggested an autocrine/paracrine role of estrogen in the regulation of estrous cycle in sheep.     

Expression of monocyte chemoattractant protein-1 and distribution of immune cell populations in the bovine corpus luteum throughout the estrous cycle.

This study characterizes the expression of monocyte chemoattractant protein-1 (MCP-1) and the relative distribution of immune cell populations in the bovine corpus luteum throughout the estrous cycle. Immunodetectable MCP-1 was evident in corpora lutea of cows at Days 6, 12, and 18 postovulation (Day 0 = ovulation, n = 4 cows/stage). Day 6 corpora lutea contained minimal MCP-1 that was confined primarily to blood vessels. In contrast, relatively intense staining for MCP-1 was observed in corpora lutea from Days 12 and 18 postovulation. MCP-1 was again most evident in the cells of the vasculature, but it was also observed surrounding individual luteal cells, particularly by Day 18. An increase in immunohistochemical expression of MCP-1 on Days 12 and 18 postovulation corresponded with increases in MCP-1 mRNA and protein in corpora lutea as determined by Northern blot analysis and ELISA. Monocytes and macrophages were the most abundant immune cells detected in the bovine corpus luteum, followed by CD8+ and CD4+ T lymphocytes. In all instances, Day 6 corpora lutea contained fewer immune cells than corpora lutea from Days 12 and 18. In conclusion, increased expression of MCP-1 was accompanied by the accumulation of immune cells in the corpora lutea of cows during the latter half of the estrous cycle (Days 12-18 postovulation). These results support the hypothesis that MCP-1 promotes immune cell recruitment into the corpus luteum to facilitate luteal regression. These results also raise a provocative issue, however, concerning the recruitment of immune cells several days in advance of the onset of luteal regression.     

Unfolding protein response signaling is involved in development,maintenance, and regression of the corpus luteum during the bovine estrous cycle

The corpus luteum (CL) is a transient endocrine organ. Development, maintenance, and regression of CL are effectively controlled by dynamic changes in gene expression. However, it is unknown what types of gene are affected during the CL life span of the estrous cycle in bovine. Here, we determined whether unfolded protein response (UPR) signaling via eIF2α/ATF4/GADD34, p90ATF6/p50ATF6, and IRE1/XBP1, which is a cellular stress response associated with the endoplasmic reticulum (ER), is involved in the bovine CL life span. Our results indicated that expression of Grp78/Bip, the master UPR regulator, was increased during the maintenance stage and rapidly decreased at the regression stage. Additionally, UPR signaling pathways genes were found to be involved in luteal phase progression during the estrous cycle. Our findings suggested that Grp78/Bip, ATF6, and XBP1 act as ER chaperones for initiating CL development and maintaining the CL. In addition, we investigated whether ER stress-mediated apoptosis is occurred through three UPR signaling pathways in CL regression stage. Interestingly, pIRE1 and CHOP were found to be involved in both the adaptive response and ER stress-mediated apoptosis. During the CL regression stage, increased expression of pJNK and CHOP, two components of ER stress-mediated apoptotic cascades, occurred before increased level of cleaved caspase 3 were observed. The present investigation was performed to identify a functional link between UPR signaling and CL life span during the bovine estrous cycle. Taken together, results from this study demonstrated that UPR protein/gene expression levels were different at various stages of the bovine CL life span. Variations in the expression of these protein/genes may play important roles in luteal stage progression during the estrous cycle.     

Expression of vascular endothelial growth factor and its receptors in the porcine corpus luteum during the estrous cycle and early pregnancy

The present study was undertaken to determine the expression of vascular endothelial growth factor (VEGF) and its receptors, fms-like tyrosine kinase (Flt-1) and fetal liver kinase-1/kinase insert domain-containing receptor (Flk-1/KDR), in the porcine corpus luteum (CL) during the estrous cycle and early pregnancy. Immunohistochemical studies localized proteins of VEGF ligand-receptor system in the cytoplasm of luteal cells and in some blood vessels. Western blot analysis revealed significantly higher levels of VEGF protein during early and mid-luteal phase (vs. late luteal phase; P<0.001 and P<0.01, respectively). Quantification of VEGF mRNA in the CL showed increased mRNA levels during entire luteal phase (vs. Days 16-17; P<0.05). Expression of Flt-1 protein remained high during luteal phase (P<0.001), but the mRNA levels tended to increase from the early to the late luteal phase. Elevated protein expression of Flk-1/KDR was found in the mid-luteal phase (vs. Days 16-17; P<0.05). However, induction of Flk-1/KDR mRNA expression occurred earlier, in early luteal phase. The lowest VEGF, Flt-1 and Flk-1/KDR mRNA and protein levels were observed in regressed CL (P<0.001). During pregnancy, VEGF, Flt-1 and Flk-1/KDR mRNA and protein expression was comparable to the mid-luteal phase. In conclusion, the present study has demonstrated dynamic expression of VEGF and its receptors in the porcine CL during the estrous cycle and early pregnancy. These data suggest that the VEGF ligand-receptor system may play an important role in the development and maintenance of the CL in pigs.     

Expression of major histocompatibility complex antigens on the bovine corpus luteum during the estrous cycle, luteolysis, and early pregnancy.

Treatment of cultured bovine luteal cells with the cytokine, interferon-gamma, induces the expression of Class II major histocompatibility complex antigens (MHC Ags). To determine if Class II MHC Ags are present on the CL in vivo and if the degree of Ag expression changes during luteal life span, bovine corpora lutea were obtained on Day 6, Days 10-12, and Day 18 of the estrous cycle and MHC Ag expression was evaluated via indirect immunofluorescence. Flow cytometry was used to determine the percentage of MHC Ag-positive cells on cell populations distinguished by cell size and intracellular density. Minimal Class II MHC Ag expression was detected on Day 6 CL (approximately 25%), which consisted primarily of smaller cells. The midcycle and late CL consisted of these small cells (SC) and two populations of large cells that differed in intracellular density, or right-angle light scatter. In midcycle CL, few (less than 25%) SC or large, dense cells (LDC) expressed the Class II MHC Ag whereas a high percentage (75%) of the large, less-dense cells (LLDC) were Class II MHC Ag-positive. Class II MHC Ag expression remained negligible on the LDC of the Day 18 CL; however, there was an elevation in the percentage of SC and LLDC expressing Class II Ag (p less than 0.05). To determine if Class II MHC Ag expression also varied with different functional states of the CL, bovine CL were collected after prostaglandin (PG) F2 alpha-induced regression and on Day 18 of early pregnancy. When luteolysis was allowed to progress in vivo, the percentage of Class II MHC Ag-positive cells was increased in all cell populations (p less than 0.05). Class II MHC Ag expression was significantly lower (p less than 0.05) on the three cell populations comprising the CL of pregnancy as compared to the Day 18 cyclic CL. It is hypothesized that enhanced expression of Class II MHC Ags on the late CL and during PGF2 alpha-induced regression may potentiate immune response mechanisms for luteolysis.