Cytokeratin expression in bovine corpora lutea

Cytokeratin (CK)-positive cells were obtained from bovine corpora lutea. When cultured, these cells behave like CK-positive endothelial cells obtained from bovine large blood vessels. The origin of CK-positive cells has now been studied in 45 bovine corpora lutea of different estrous cycle stages. Additionally, 7 corpora lutea of pregnant cows were examined. The tissues were grouped into early stage (days 2 to 4), secretory stage (days 5 to 17) and late stage (days 18 to 21) according to gross morphology, wet weight and total progesterone content. One portion of a corpus luteum was used for immunohistochemistry, and another for Western blot analysis. Twenty-six of the 45 corpora lutea showed CK expression, as confirmed by immunostaining and Western blotting. Cytokeratin expression was found in all corporalutea from the early stage, in 14 of 26 corpora lutea from the secretory stage, and 3 of 10 from the late stage. Early stage corpora lutea displayed zonation such that a high number of CK-positive luteal cells occurred in the region of the previous granulosa layer and a very low number in the previous thecal layer. Secretory CK-positive corpora lutea showed uniformly distributed, predominantly large luteal cells. In secretory corpora lutea of group A, CK-positive cells and a distinct microvascular tree were seen, the latter visualized by factor VIII-related antigen immunolabelling of endothelial cells. Group B showed none or very few CK-positive cells. Corpora lutea of pregnant cows behaved like corpora lutea of group B. Roughly 1% of CK-positive cells closely associated with the capillary wall were sometimes reminiscent of endothelial cell sprouts.     

Quantitative cell composition of human and bovine corpora lutea from various reproductive states

The cell composition of human and bovine corpora lutea (CL) from various reproductive states was investigated by computerized video-based interactive Bioquant image analysis system IV and by light microscope immunocytochemistry. Human and bovine CL contained more nonluteal cells than luteal cells. Human CL contained a lower number of luteal and a greater number of nonluteal cells than bovine CL. Regardless of the reproductive state, human CL contained more small luteal cells than large luteal cells. In all reproductive states except in the late luteal phase, the bovine CL also contained more small luteal cells than large luteal cells. The average sizes of all the cells in human CL were smaller than in bovine CL. Human CL contained more vascular space than bovine CL during mid and late luteal phases. The number of luteal cells increased and nonluteal cells decreased from early to mid luteal phase, and then luteal cells decreased and nonluteal cells increased in late luteal phase and in degenerating human and bovine CL. While the change of number of small and large luteal cells first occurred from early to mid luteal phase in human CL, it did not take place until the late luteal phase in bovine CL. The average size of large luteal cells in humans and of small luteal cells in cattle did not change, whereas size of the other cells changed in different reproductive states in both human and bovine CL. The cell composition of term pregnancy human CL was similar to mid or late luteal phase, whereas the cell composition of early pregnancy bovine CL was similar to mid luteal phase.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Immunocytochemical localization of relaxin in human corpora lutea: cellular and subcellular distribution and dependence on reproductive state

Relaxin is one of the hormones present during pregnancy and it is synthesized primarily by corpora lutea (CL). Other reproductive tissues including CL of the menstrual cycle may also synthesize this hormone. Very little is known, however, about the cellular and subcellular distribution of relaxin in human CL and dependence of luteal relaxin on the reproductive state. The light and electron microscope immunocytochemical studies described here were undertaken to obtain this information using antisera to porcine and human relaxin. Immunostaining was found in large luteal cells (17-30 microns) but not in small luteal cells (7-16 microns) or in nonluteal cells in any of the reproductive states or in human hepatocytes. Luteal immunostaining was low in early luteal phase; it increased progressively, reaching the highest level in late luteal phase, and then decreased greatly in corpora albicantia. Term pregnancy CL contained similar immunostaining as early luteal phase CL. Mid luteal phase CL contained more immunostained cells than late luteal phase CL, but the late luteal phase CL contained a greater amount of immunostaining per cell than mid luteal phase CL. The immunogold particles due to relaxin were primarily present in secretory granules and to a small extent in rough endoplasmic reticulum. Quantitation revealed that secretory granules contained a much higher number of gold particles than did rough endoplasmic reticulum. These two organelles from late luteal phase CL contained greater numbers of gold particles than those from mid luteal phase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Total ascorbate and dehydroascorbate concentrations in porcine ovarian stroma, follicles and corpora lutea throughout the estrous cycle and pregnancy

Ovarian tissues are thought to require ascorbate as an antioxidant and enzymatic cofactor for the processes of steroid and collagen synthesis. We measured the concentrations of total ascorbate and oxidized ascorbate (dehydroascorbate, DHA) in ovarian stroma, follicles and corpora lutea (CL) throughout the estrous cycle and pregnancy of the sow. Both total ascorbate and DHA concentrations were greatest in luteal tissue and lowest in ovarian stroma across all stages examined. Within the CL, total ascorbate levels were lowest during the early, early-mid, and late luteal phase and were elevated during the mid-luteal phase. Luteal total ascorbate concentrations were further elevated during early pregnancy and were comparable to mid-luteal phase concentrations during the remainder of gestation. Luteal DHA concentrations decreased from mid to late luteal phase, and were elevated throughout pregnancy. As the CL aged during the cycle, the DHA/total ascorbate ratio decreased and remained low throughout pregnancy. Total ascorbate concentrations in follicular tissue increased during the follicular phase and were lowest during the early luteal phase. The DHA concentrations and DHA/total ascorbate ratios in follicular tissue did not differ with stage. Total ascorbate and DHA concentrations in ovarian stroma were low and did not vary with stage. We conclude that periods of maximal luteal and follicular function are associated with increased concentrations of total ascorbate within the tissue. Furthermore, luteolysis appears to be associated with depletion of luteal ascorbate species.     

Changes in activities of superoxide dismutase, nitric oxide synthase, glutathione-dependent enzymes and the incidence of apoptosis in sheep corpus luteum during the estrous cycle

Anti-oxidative enzymes play a role in protecting cells from oxidative stress-induced cell death. The present study was conducted to evaluate whether the anti-oxidant and pro-oxidant enzymatic capacities of the sheep corpus luteum (CL) are correlated with steroidogenic and structural status of the gland during the estrous cycle. Steroidogenic activity, apoptosis and superoxide dismutase (SOD1 and SOD2), nitric oxide synthase (NOS), glutathione peroxidase (GPX), glutathione reductase (GSR) and glutathione S-transferase (GST) activities were determined in the CL at specific developmental stages of the luteal phase. The intensity of apoptotic DNA fragmentation, characteristic of physiological cell death, was much greater in CL at late luteal phase than at early and mid-luteal phase, concomitantly with the diminution in the plasma progesterone concentrations from mid-to late luteal phase. SOD1 and GPX activities increased from early to mid-luteal phase, and increased further at late luteal phase. SOD2 and GST activities were not different between early and mid-luteal phase, but increased at late luteal phase. GSR activity was not different between any luteal phase examined. NOS activity decreased from early to mid- and late luteal phase. These results show that the activities of SOD1, SOD2, NOS, GPX, GSR and GST in the sheep CL are subject to major changes during the estrous cycle, and that the anti-oxidant and pro-oxidant enzymatic capacities of luteal cells are not correlated with cell steroidogenic status and integrity during the late luteal phase.     

Receptors for luteinizing hormone and follicle-stimulating hormone in largest follicles of postpartum beef cows

Follicles collected from cows destined to enter relatively normal or short luteal phases if induced to ovulate were compared for numbers of receptors for luteinizing hormone (LH) in granulosal and thecal cells and for follicle-stimulating hormone (FSH) in granulosal cells. Eleven suckled beef cows received ear implants of 6 mg norgestomet for 9 days (expected normal luteal phase) and 11 served as controls (expected short luteal phase). At 48 h after implants were removed (average 34 days postpartum), the ovary containing the largest follicle was identified by transrectal ultrasound and removed. The largest follicle was dissected free of surrounding ovarian stroma and frozen in liquid nitrogen. Thecal and granulosal cells were isolated, and numbers of receptors for LH and FSH in granulosal cells and for LH in thecal cells were quantified. Concentrations of estradiol were measured in follicular fluid. Both granulosal and thecal cells from norgestomet-treated cows had greater numbers of receptors for LH than did those from control cows (p less than 0.01). Numbers of receptors for FSH in granulosal cells did not differ between treated and control cows. Follicles from norgestomet-treated cows were heavier (p less than 0.01) than follicles from control cows, mostly due to greater amounts of follicular fluid (p less than 0.01). Concentrations of estradiol were higher in follicular fluid from the treated cows (p less than 0.05). It is suggested that increases in numbers of follicular receptors for LH and secretion of estradiol are integral components of a sequence of events by which norgestomet prepares follicles to become fully functional corpora lutea.     

Progesterone and prostaglandin production by primate luteal cells collected at various stages of the luteal phase: modulation by calcium ionophore

Prostaglandins (PG) are produced by the corpus luteum (CL) of the rhesus monkey and may be involved in luteal regulation. Intracellular calcium has also been implicated as a mediator of luteolysis in domestic and laboratory species; however, its role in primate luteal function has not been investigated. The objectives of this study were to characterize temporal changes in basal and stimulated luteal PG production by CL of rhesus monkeys, and to examine the effects of calcium ionophore (CaI) on basal and gonadotropin-stimulated progesterone (P) production by the CL. CL were collected at various times after the estimated day of the luteinizing hormone (LH) surge: 5 days (early luteal phase, n = 4), 8-10 days (mid-luteal phase, n = 8), and 12-14 days (late luteal phase, n = 5). Dispersed luteal cells were incubated in the absence and presence of CaI, or with human chorionic gonadotropin (hCG) plus CaI at 37 degrees C for 8 h. PG and P concentrations in the medium were measured by radioimmunoassay. PGE2 and 6-keto-PGF1 alpha production decreased (p less than 0.05) from early luteal phase to mid-luteal phase and remained lower (p less than 0.05) during late luteal phase for all treatment groups. PGF2 alpha production decreased (p less than 0.05) from early to mid-luteal phase and rebounded in late luteal phase to the same level (p greater than 0.05) found in early luteal phase. CaI stimulated (p less than 0.05) basal PG production. The degree of stimulation was similar throughout the luteal phase (p greater than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence for a switch in the site of relaxin production from small theca-derived cells to large luteal cells during early pregnancy in the pig

The presence of immunoreactive relaxin was studied in corpora lutea of sows during the oestrous cycle and early pregnancy by immunohistochemistry and radioimmunoassay using three different anti-relaxin sera. Sections were immunostained using the peroxidase-anti-peroxidase or the immunogold-silver technique. Before Day 14, staining in corpora lutea from non-pregnant and pregnant animals was indistinguishable. With all antisera, no immunostaining was seen on Day 3, but was detected on Days 5-7 in cells from the theca interna. In non-pregnant animals, this immunostaining decreased and by Day 15 only an occasional large cell in the centre of the corpus luteum was stained. No staining was seen by Day 22. The relaxin content of corpora lutea measured by radioimmunoassay remained low throughout the luteal phase. In contrast, the amount of immunoreactive relaxin in corpora lutea rose dramatically (140-fold) between Days 11 and 14 of pregnancy and by Day 14 of pregnancy immunostaining was seen in the majority of large luteal cells. By Day 20 of pregnancy the concentrations of immunoreactive relaxin had further increased. Histochemical staining for alkaline phosphatase suggested that, while the relaxin-immunoreactive cells seen in the early luteal phase may be theca-derived, those during early pregnancy may be derived from the granulosa. The results are compatible with the suggestion that relaxin is produced by theca-derived cells during the early luteal phase and that between Days 11 and 14 there is a switch in the site of relaxin synthesis from theca-derived cells to granulosa-derived large luteal cells. In the absence of luteolysis, as during pregnancy, this switch is accompanied by a dramatic increase in relaxin synthesis.     

Cellular distribution and cycle phase dependency of gonadotropin and eicosanoid binding sites in bovine corpora lutea.

Bovine luteal functions are regulated by gonadotropins and eicosanoids. The specific binding sites that presumably mediate the actions of these regulatory agents have previously been characterized in bovine luteal tissue. However, the cellular distribution and/or the cycle phase dependency of these binding sites have never been investigated. In the present study, we investigated these parameters by using quantitative light microscope autoradiography. The results showed that both small and large luteal cells contained binding sites for LH/hCG, prostaglandin (PG)E2, PGF2 alpha, PGI2, and leukotriene (LT)C4. In addition, luteal blood vessels contained LH/hCG and LTC4 binding sites and luteal fibroblasts contained PGE2 binding sites. On a per cell basis, there were more binding sites for all ligands in large luteal cells as compared to small or nonluteal cells. After correction for the cellular area differences, small luteal cells contained more LH/hCG, PGE2, PGI2, and LTC4 binding sites, while large luteal cells contained more PGF2 alpha binding sites. The small and large luteal cell binding of hCG, PGE2, PGI2, and LTC4 increased from early to mid luteal phase, followed by a decline in the late luteal phase. PGF2 alpha binding, on the other hand, increased from early to late luteal phase. In contrast to luteal cells, binding of hCG and LTC4 to luteal blood vessels and binding of PGE2 to luteal fibroblasts did not change during the cycle. These results suggest that LH/hCG and eicosanoid regulation of luteal function is more complex than previously envisioned and it involves both small and large luteal cells and, in some cases, also nonluteal cells.     

Effect of prostaglandin E2 and tumor necrosis factor alpha on the VEGF-receptor system expression in cultured porcine luteal cells

The purpose of the present study was to investigate the effects of prostaglandin (PG) E(2) and tumor necrosis factor (TNF) alpha on 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 cultured porcine luteal cells. Real-time PCR was used for quantification of VEGF and its receptors mRNA, whereas VEGF release by luteal cells was determined by radioimmunoassay (RIA). Only the highest dose of PGE(2) (1 microM) after 6 hr of incubation stimulated VEGF release by luteal cells collected in the mid-luteal phase (P < 0.05). Moreover, PGE(2) (100 nM, 1 microM) significantly stimulated VEGF secretion by luteal cells in the late phase and during pregnancy on Days 10-12 (P < 0.05). Elevated mRNA expression of both VEGF 120 and VEGF 164 isoforms was found in luteal cells cultured with PGE(2). The lack of an effect of PGE(2) on VEGF receptors mRNA expression was observed. TNFalpha was able to significantly stimulate VEGF release from cells obtained in the mid- and late luteal phase or during early pregnancy. All tested doses enhanced mRNA levels of VEGF 120 isoform, but not VEGF 164. Additionally, TNFalpha was able to decrease Flk-1/KDR mRNA expression, whereas Flt-1 mRNA levels were not affected. These results indicated that PGE(2) and TNFalpha influenced VEGF ligand-receptor system expression in porcine luteal cells and may therefore play an important role in regulation of luteal functions during the estrous cycle and pregnancy in pigs.     

Multiparametric evaluation of flow cytometric synthesis phase fraction determination in dual-labelled breast carcinomas.

Multiparametric, two-color DNA and cell cycle analyses were performed on 112 consecutive mechanically dissociated, ethanol-fixed breast carcinomas using a dual-label method with monoclonal antibodies (CAM 5.2) to cytokeratin (CK) and leukocyte common antigen (LCA) with propidium iodide (PI) staining. There was marked intertumoral variation of CK-positive (range, 3-87%; mean, 40%) and LCA-positive (range, 1-28%; mean, 6.5%) events in DNA histograms. Approximately 70% of DNA aneuploid cells were CK positive. CAM 5.2-stained (avidin-biotin technique) Cytospin preparations correlated with flow cytometric (FCM) detection of CK-positive cells in 15/21 (71%) cases. In each discrepant case, FCM detected greater numbers of CK-positive cells. Cytospin controls of tumor suspensions revealed that cytoplasmic loss was the major cause of decreased CK staining. Synthesis phase fraction (SPF) calculation from CK-gated histograms resulted in kinetic indices (mean ungated, 12.3%, vs. mean CK-gated, 16.8%; P less than .01) with improved statistical correlations with tumor grade and estrogen receptor (ER) status. Differences between ungated vs. CK-gated SPF were greatest in cases having less than 20% CK-positive events (P less than .05). Cases with lower CK staining events generally had higher SPF and were more often high grade (below median CK staining, 61% high grade, vs. above median CK staining, 31% high grade) and ER-negative (below median CK staining, 55% ER negative, vs. above median CK staining, 12% ER negative).(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression of cathepsin K mRNA and protein in odontoclasts after experimental tooth movement in the mouse maxilla by in situ hybridization and immunoelectron microscopy

This study demonstrated the simultaneous expression of cathepsin K (CK) mRNA by in situ hybridization and CK protein by immunoelectron microscopy in odontoclasts in mouse maxillae after experimental tooth movement. On the pressure side (the area under pressure during tooth movement), CK mRNA was detected in odontoclasts in resorption lacunae in the tooth root, in osteoclasts in bone resorption lacuane, and in fibroblasts in the periodontal ligament. Using electron microscopy, CK protein was detected at the apex of odontoclasts, intracellularly in vesicles and granules, and extracellularly in irregularly shaped vacuoles (extracellular spaces), on the plasma membrane of the ruffled border, and on and between typical striated type I collagen fibrils in the lacunae. These vesicles and granules appeared to fuse with irregular vacuoles containing CK-positive fragmented fibril-like structures close to the ruffled border. In the basolateral portion of odontoclasts, small amounts of CK-positive rough endoplasmic reticulum (ER) were found. CK-positive intracellular vacuoles (not extracellular spaces) also appeared to fuse with the vesicles and granules. However, these fused organelles rarely contained fragmented fibril-like structures. They are probably endolysosomes. The distribution of CK in odontoclasts was similar to that previously seen in osteoclasts. Furthermore, CK-positive fibril-like structures were found in the vacuoles of fibroblasts. These results indicated that during tooth movement CK is synthesized in odontoclasts on the pressure side and secreted into the tooth resorption lacunae. Therefore, CK may take part in the degradation of the dentin matrix (type I collagen fibrils and non-collagenous protein) of the tooth root, and in the subsequent intracellular degradation of endocytosed fragmented fibril-like structures in endolysosomes.     

The enzymes in cyclooxygenase and lipoxygenase pathways of arachidonic acid metabolism in human corpora lutea: dependence on luteal phase, cellular and subcellular distribution

Eicosanoids synthesized within corpus luteum are presumed to regulate luteal function in women. However, the potential cellular source(s) of the eicosanoids, whether small and large luteal cells differ in eicosanoid synthesis and whether eicosanoids other than prostaglandin (PG)E2, PGF2 alpha and 6-keto-PGI1 alpha can be synthesized, have not been investigated. The present immunocytochemical studies were undertaken to answer these questions using mono and polyclonal antibodies to several enzymes in arachidonic acid metabolism by cyclooxygenase and lipoxygenase pathways. Human corpora lutea from early (n = 5), mid (n = 6) and late (n = 3) luteal phases were specifically immunostained for all the enzymes. All the enzymes were present in small and large luteal cells as well as in non luteal cells. However, small luteal cells contained more immunoreactive 5-lipoxygenase, PGD2 and PGF2 alpha synthases; large luteal cells contained more TXA2 synthase and 12-lipoxygenase; small and large luteal cells contained similar amounts of cyclooxygenase and PGI2 synthase. In all the cells, immunoreactive PGD2, PGI2 and TXA2 synthases increased from early to mid luteal phase and then declined in late luteal phase. Cyclooxygenase, 5- and 12-lipoxygenases and PGF2 alpha synthase, on the other hand, increased from early to mid and mid to late luteal phases. Immunoreactive cyclooxygenase and 5- and 12-lipoxygenases were present primarily in rough endoplasmic reticulum (ER) and/or smooth ER and cytoplasm. Quite unexpectedly, all three enzymes were also found in nuclear membranes, condensed chromatin and especially at the perimeter of condensed chromatin. Dispersed chromatin contained very little or no immunoreactive enzyme. These results indicate that regulation of human luteal function by eicosanoids synthesized within the corpus luteum is complex involving perhaps a) small and large luteal as well as non luteal cells, b) eicosanoids which have not been previously considered to play a role in luteal function and c) coordinate regulation of more than one enzyme in the pathways of arachidonic acid metabolism.     

Gap junctional intercellular communication of bovine luteal cells from several stages of the estrous cycle: Effects of prostaglandin F2α, protein kinase C and calcium

Cellular interactions mediated by both contact-dependent and contact-independent mechanisms are probably important to maintain luteal function. The present studies were performed to evaluate the effects of luteotropic and luteolytic hormones, and also intracellular regulators, on contact-dependent gap junctional intercellular communication (GJIC) of bovine luteal cells from several stages of luteal development. Bovine corpora lutea (CL) from the early, mid and late luteal phases of the estrous cycle were dispersed with collagenase and incubated with no treatment, LH, PGF or LH + PGF (Experiment 1), or with no treatment, or agonists or antagonists of protein kinase C (TPA or H-7) or calcium (A23187 or EGTA; Experiment 2). After incubation, media were collected for determination of progesterone concentrations. Then the rate of GJIC was evaluated for small luteal cells in contact with small luteal cells, and large luteal cells in contact with small luteal cells by using the fluorescence recovery after photobleaching technique and laser cytometry. Luteal cells from each stage of the estrous cycle exhibited GJIC, but the rate of GJIC was least (P<0.05) for luteal cells from the late luteal phase. LH increased (P<0.05) GJIC between small luteal cells from the mid and late but not the early luteal phase. PGF increased (P<0.05) GjIC between small luteal cells from the mid luteal phase and diminished (P<0.05) LH-stimulatory effects on GjIC between small luteal cells from the late luteal phase. Throughout the estrous cycle, TPA decreased (P<0.05) the rate of GjIC between large and small, and between small luteal cells, and A23187 decreased (P<0.05) the rate of GJIC between large and small luteal cells. LH and LH + PGF, but not PGF alone increased (P<0.05) progesterone secretion by luteal cells from the mid and late luteal phases. Agonists or antagonists of PKC or calcium did not affect progesterone secretion by luteal cells. These data demonstrate that both luteal cell types communicate with small luteal cells, and the rate of communication depends on the stage of luteal development. LH and PGF affect GjIC between small luteal cells during the fully differentiated (mid-luteal) and regressing (late luteal) stages of the estrous cycle. In contrast, at all stages of luteal development, activation of PKC decreases GjIC between small and between large and small luteal cells, whereas calcium ionophore decreases GjIC only between large and small luteal cells. Luteotropic and luteolytic hormones, and intracellular regulators, may be involved in regulation of cellular interactions within bovine CL which likely is an important mechanism for coordination of luteal function.     

The presence of pregnancy-associated plasma protein-A in human corpora lutea: cellular and subcellular distribution and dependence on reproductive state.

Pregnancy-associated plasma protein-A (PAPP-A) is a high-molecular-weight glycoprotein primarily secreted by syncytiotrophoblasts of human placenta. It is not known, however, whether human CL of menstrual cycle or pregnancy also contain this protein. Therefore, light and electron microscope immunocytochemical studies were undertaken to investigate the presence, cellular and subcellular distribution, and dependence of luteal PAPP-A content on reproductive state. Human CL from early, mid, and late luteal phases and from term pregnancies immunostained specifically for PAPP-A. Immunostaining was found in large luteal cells (17-30 microns) but not in small luteal cells (7-16 microns) or in nonluteal cells in any of the reproductive states. Immunostaining was not found in negative control tissues, i.e. human liver or bovine CL of pregnancy. As expected however, term-pregnancy human placenta used for a positive control tissue immunostained intensely for PAPP-A. The luteal immunostaining was highest in early luteal phase, decreased progressively from early to mid and from mid to late luteal phases, and then disappeared in corpora albicantia. The relative intensity of immunostaining in early luteal phase human CL was similar to that in term-pregnancy human placenta and higher than in term-pregnancy human CL. The immunogold particles due to PAPP-A were primarily associated with secretory granules of large luteal cells. A small number of gold particles were also found in rough endoplasmic reticulum and cytoplasm. In conclusion, human CL contain immunoreactive PAPP-A. The luteal content varies with reproductive state, with the highest amount found in early luteal phase CL.(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression of mRNA encoding insulin-like growth factors I and II and the type 1 IGF receptor in the bovine corpus luteum at defined stages of the oestrous cycle

Previous studies have implicated insulin-like growth factors I and II (IGF-I and -II), in the regulation of ovarian function. The present study investigated the localization of mRNA encoding IGF-I and -II and the type 1 IGF receptor using in situ hybridization to determine further the roles of the IGFs within the bovine corpus luteum at precise stages of the oestrous cycle. Luteal expression of mRNA encoding IGF-I and -II and the type 1 IGF receptor was detected throughout the oestrous cycle. The expression of IGF-I mRNAvaried significantly during the oestrous cycle. IGF-I mRNA concentrations were significantly higher on day 15 than on day 10, and IGF-I mRNA in the regressing corpus luteum at 48 h after administration of exogenous prostaglandin was significantly greater than in the early or mid-luteal phase (days 5 and 10). In contrast, there was no significant effect of day of the oestrous cycle on expression of mRNA for IGF-II and the type 1 IGF receptor in the corpus luteum. Expression of IGF-II mRNA was localized to a subset of steroidogenic luteal cells and was also associated with cells of the luteal vasculature. mRNA encoding the type 1 IGF receptor was widely expressed in a pattern indicative of expression in large and small luteal cells. These data demonstrate that the bovine corpus luteum is a site of IGF production and reception throughout the luteal phase. Furthermore, this study highlights the potential of IGF-II in addition to IGF-I in the autocrine and paracrine regulation of luteal function.     

Asymmetric secretion of principal ovarian venous steroids in the primate luteal phase

We have characterized the degree of asymmetry of ovarian steroid secretion in the luteal phase of the menstrual cycle in rhesus and cynomolgus monkeys. Femoral blood levels of FSH, LH, progesterone, estradiol and 17-hydroxyprogesterone were determined. In addition, laparotomies were performed in the early, mid or late luteal phase to facilitate localization of the corpus luteum and collection of ovarian venous blood. We conclude that: 1) the ovary bearing the active corpus luteum contributes virtually all of the progesterone entering peripheral circulation in the luteal phase; 2) the ipsilateral ovary secretes more 17-hydroxyprogesterone than the contralateral one, although both are active in the luteal phase; and 3) the asymmetrical secretion of estradiol was manifest only in the early and mid-luteal phase, with ovarian symmetry being reestablished in the late luteal phase.