Production of heparin-binding angiogenic factor(s) by bovine corpora lutea during pregnancy.

Effects of luteal-conditioned media (LCM) on proliferation and migration of endothelial cells were used to assess angiogenic activity of corpora lutea (CL) obtained from cows on d 100 (n = 5), 150 (n = 6), 200 (n = 6), and 250 (n = 6) of gestation. Explants of CL (200 mg) were incubated for 6 h in 3 mL of serum-free media containing no hormone, LH (1 microgram/mL), prostaglandin F2 alpha (PGF2 alpha; 3 microM), or both hormones. Media from the four stages of gestation were subjected to the following procedures: 1) ultrafiltration, 2) high-salt (3.0 M NaCl) treatment and then ultrafiltration, 3) heat treatment, 4) heparin-Sepharose affinity chromatography, 5) immunoneutralization with specific antibodies against heparin-binding growth factor (HBGF)-1 and against HBGF-2, and 6) dot immunoblot assay for HBGF-2. Fractions from the first five procedures were evaluated in the endothelial cell proliferation bioassay. In addition, progesterone concentration of LCM was determined by RIA. Across all days of gestation and hormone treatments, LCM stimulated (P less than .05) proliferation and migration of endothelial cells, but activities did not differ among stages of gestation or hormone treatments. Both mitogenic and migration-stimulating fractions seemed to have Mr greater than 100,000. The mitogenic activity fraction had an apparent Mr greater than 100,000 even after treatment with high salt and was heat-labile. This endothelial mitogen was retained on heparin-Sepharose columns and was eluted with 2.0 M NaCl. Mitogenic activity was partly neutralized (P less than .05) by antibodies against HBGF-2 but not HBGF-1. Presence of HBGF-2 in LCM was detected by dot immunoblot assay.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chemoattractant properties of conditioned medium from equine corpora lutea collected at various stages of the oestrous cycle

This study investigated the chemotactic activity of equine CL at different stages of the oestrous cycle. The purpose of this was to ascertain whether luteal tissue itself contributes to the massive influx of leucocytes around the time of natural and induced luteal regression. Corpora lutea were collected at different stages of dioestrus and after treatment with PGF2alpha. Culture medium harvested after incubation of luteal tissue for 20 h was chemotactic for both polymorphonuclear and mononuclear cells in late dioestrus (before functional regression) as well as after natural and induced luteal regression. By contrast, midluteal tissue showed no chemotactic activity. This is the first report of the ability of equine luteal tissue actively to recruit inflammatory cells in vitro and supports our earlier findings that this infiltration starts prior to functional luteolysis. We hypothesise that this early influx of inflammatory cells may play an active role in luteal regression. Further research is needed to identify the specific chemotactic factor(s).     

The localization and expression of epidermal growth factor and epidermal growth factor receptor in bovine ovary during oestrous cycle

Epidermal growth factor (EGF) is one of the important regulatory factors of EGF family. EGF has been indicated to effectively inhibit the apoptosis of follicular cells, to promote the proliferation of granulosa cells and the maturation of oocytes, and to induce ovulation process via binding to epidermal growth factor receptor (EGFR). However, little is known about the distribution and expression of EGF and EGFR in cattle ovary especially during oestrous cycle. In this study, the localization and expression rule of EGF and EGFR in cattle ovaries of follicular phase and luteal phase at different time points in oestrous cycle were investigated by using IHC and real-time qPCR. The results showed that EGF and EGFR in cattle ovary were mainly expressed in granulosa cells, cumulus cells, oocytes, zona pellucida, follicular fluid and theca folliculi externa of follicles. The protein and mRNA expression of EGF/EGFR in follicles changed regularly with the follicular growth wave both in follicular and in luteal phase ovaries. In follicular phase ovaries, the protein expression of EGF and EGFR was higher in antral follicles than that of those in other follicles during follicular growth stage, and the mRNA expression of EGFR was also increased in stage of dominant follicle selection. However, in luteal phase ovaries, the growth of follicles was impeded during corpus luteum development under the action of progesterone secreted by granular lutein cell. The mRNA and protein expressions of EGF and EGFR in ovarian follicles during oestrous cycle indicate that they play a role in promoting follicular development in follicular growth waves and mediating the selection process of dominant follicles.     

Uterine motility of the sow during the oestrous cycle and early pregnancy

With electromyography and strain gauges the uterine motility of the sow during the oestrous cycle and early pregnancy was studied. Special attention was paid to characterization of myometrial activity at the time of intrauterine migration of blastocysts.From recordings of 4 animals (3 became pregnant) 3 types of electrical spiking activity (each could be correlated with an elevation of the strain gauge tension) were discerned. Two of them appeared regularly and were suitable for analysis: High Voltage Slow Acticity (with relative high amplitude and low frequency of spikes) and Low Voltage Fast Activity (with relative low aplitude and high frequency of spikes).The sexual status markedly influenced myometrial activity: during prooestrus and oestrus only one type of electrical activity was present whereas the myoelectrical complex (an episode of this activity and the subsequent interval of inactivity) was shorter than 10 min. During dioestrus the myoelectrical complex was longer than 10 min and High Voltage Slow Activity (solely on the cervix and bifurcation) and Low Voltage Fast Activity occurred simultaneously in episodes which mainly appeared to originate on the bifurcation.The characteristics of uterine activity during pregnancy were similar to those of a cyclic sow until day 12. It was only on day 12 that gestation appeared from an increased frequency of myoelectrical complexes.It is concluded that Low Voltage Fast Activity as it was found on the uterine horn at days 8–9 might be involved in the process of intra-uterine migration of blastocysts. In cyclic and in pregnant animals the patterns of Low Voltage Fast Activity were similar. Therefore, the occurrence of Low Voltage Fast Activity is independent of the actual presence of blastocysts. It seems to be exhibited in dependence on the ovarian hormones.     

Androgen receptor mRNA expression in the bovine ovary

Previous studies have shown that androgen receptor (AR) is expressed in granulosa cells of healthy, growing ovarian follicles in rats and primates. However, AR expression in the bovine ovary has not been examined. Therefore, a 346-base pair segment of the bovine AR was cloned and sequenced. Using a ribonuclease protection assay, AR expression was detected in total RNA from bovine ovarian cortex. Expression (absence or presence) of AR mRNA was detected by in situ hybridization in bovine ovarian cortex. Follicles (n = 32) were classified as follows: type 1 (1 layer of flattened granulosa cells), type 2 (1-1.5 layers of cuboidal granulosa cells), type 3 (2-3 layers of granulosa cells), type 4 (4-6 layers of cuboidal granulosa cells and formation of thecal layer), and type 5 (>6 layers of cuboidal granulosa cells, defined theca layer, and antrum formation). Frequency of AR mRNA expression increased (P < 0.001) as follicles entered the growing pool. Expression of AR mRNA was absent in type 1 follicles (n = 8), but present in the granulosa cells of 41% of type 2 follicles (n = 12). In types 3-5 follicles, AR mRNA expression was present in granulosa cells of 100% of follicles examined (n = 4, 4, and 4, respectively) and was greater than type 1 follicles (P = 0.002). These data provide evidence of AR mRNA expression in bovine follicles and suggest that AR mRNA increases during early follicle development.     

Microvascularization and angiogenic activity of equine corpora lutea throughout the estrous cycle

Corpus luteum growth and endocrine function are closely dependent on the formation of new capillaries. The objectives of this study were to evaluate (i) tissue growth and microvascular development in the equine cyclic luteal structures; (ii) in vitro angiogenic activity of luteal tissues in response to luteotrophic (LH, PGE₂) and luteolytic (PGF₂) hormones and (iii) to relate data to luteal endocrinological function. Our results show that microvascular density was increased in the early and mid luteal phase, followed by a fall in the late luteal phase and a further decrease in the corpus albicans. Hyperplasia of luteal tissue increased until the mid luteal phase and it was followed by tissue regression. Luteal explants were cultured with no hormone added, or with PGF₂, LH, PGE₂, LH + PGE₂ or LH + PGF₂. Media conditioned by equine luteal tissue from different stages of the luteal phase were able to stimulate mitogenesis of bovine aortic endothelial cells (BAEC), suggesting the presence of angiogenic activity. No difference was observed among luteal structures on their mitogenic capacity, for any treatment used. Nevertheless, Late-CL conditioned-media with PGF₂ showed a significant decrease in BAEC proliferation (p < 0.05) and LH + PGF₂ a tendency to reduce mitogenesis. Thus, prostaglandin F₂ may play a role on vascular regression of the CL during the late luteal phase in the mare. These data suggest that luteal angiogenesis and vascular regression in the mare are coordinated with the development of non-vascular tissue and might be regulated by many different factors.     

Luteinizing hormone receptor number and affinity in corpora lutea from prepuberal gilts induced to ovulate and spontaneous corpora lutea of mature gilts

This study was designed to determine if luteal cell receptors for luteinizing hormone/human chorionic gonadotropin (LH/hCG) contribute to the previously demonstrated abnormal function of induced corpora lutea (CL) in gilts. Twenty-five prepuberal (P) gilts, induced to ovulate with 1,500 IU pregnant mare serum gonadotropin followed 72 h later with 500 IU hCG (d 0 = day of hCG), and 22 mature (M) gilts that had displayed two or more estrous cycles were ovariectomized (OVX) on d 10, 14, 18, 22 or 26 after the onset of estrus. All gilts except those OVX on d 10 were hysterectomized between d 6 and 9 to ensure luteal maintenance. The CL were stored at -196 degrees C until determination of LH/hCG receptor number and dissociation constant (KD) by saturation analysis. Receptor number was greater for M than for P gilts on d 14 (P less than .07) and d 18 (P less than .01). The KD was greater in M than in P gilts on d 14 (P less than .01) and d 18 (P less than .0001). The LH/hCG receptor number and KD of P gilts remained the same throughout the days studied. The LH/hCG receptor number (fmol/mg protein) of M gilts was elevated on d 10, 14, and 18 (50.8, 50.4 and 51.4, respectively) and decreased on d 22 (26.5) and d 26 (25.4) to values similar to those of P gilts. In M gilts, KD increased on d 14, remained high on d 18 and decreased on d 22. We suggest that abnormal function of induced CL in P gilts may be due to an elevated LH receptor number.     

Expression of oestrogen receptor,androgen receptor and progesterone nuclear receptor in sheep uterus during the oestrous cycle

Oestrogen, androgen and progesterone are involved in the regulation of uterine physiological functions, with the participation of the following proteins: oestrogen receptor (ER), androgen receptor (AR) and progesterone nuclear receptor (PGR). In this study, we used immunohistochemistry to detect the localization of ERα, ERβ, AR and PGR in sheep uterus. Additionally, we used real‐time polymerase chain reaction (RT‐qPCR) and Western blot technique to analyse their expression profiles at different stages of sheep oestrous cycle in the endometrium and myometrium. Immunohistochemical analysis showed that ERα, ERβ, AR and PGR were present in sheep uterus in oestrus, mainly in the uterine luminal epithelium, stroma, gland and myometrium. Real‐time polymerase chain reaction results showed that in the endometrium, ERα expression level was highest in oestrus. ERβ and PGR, instead, were highly expressed in pro‐oestrus. In the myometrium, ERα was highly expressed in both oestrus and pro‐oestrus, and ERβ was highly expressed in oestrus and dioestrus. Progesterone nuclear receptor expression was highest in oestrus, followed by metoestrus. In the endometrium, both receptors ERα and ERβ were abundant in pro‐oestrus, while the maximum AR protein content was found in oestrus. At this stage of the oestrous cycle, PGR protein concentration in the myometrium was significantly lower than those observed in other stages. These results suggest that these receptors are important for sheep reproductive function, as their expression at mRNA and protein levels exhibits particular time‐ and tissue‐specific profiles along the oestrous cycle.     

Progesterone receptor mRNA levels during pregnancy, labor, lactation and the estrous cycle in rat uterus

Progesterone plays important roles in the regulation of female reproduction. In this study, progesterone receptor (PR) mRNA levels in rat uterus during pregnancy, labor, lactation and the estrous cycle were examined by competitive RT-PCR. During pregnancy and lactation, PR mRNA levels had decreased on day 20 of pregnancy (P20) and P21 compared with P15 but increased during labor. After a decline on day 1 of lactation (L1), PR mRNA levels had increased again on L3 and L14 compared with P15, P18, P20, P21 and P21pm (at 2200-2300 h on P21). There was no significant change in the PR mRNA level during the estrous cycle. The PR mRNA level did not change during 1 week of progesterone treatment or afterwards. Injection of 17beta-estradiol did not affect PR mRNA levels in rats treated with progesterone or those without any injections. In rats on P18, 17beta-estradiol injection did not change PR mRNA levels after sham-operation but induced an increase in PR mRNA levels of rats ovariectomized 6 h before the treatment. These results suggest that uterine PR mRNA levels are differently regulated during late pregnancy, labor and lactation, and during labor estrogen is one of the essential factors for the increase in PR mRNA levels.     

Characterization of mesenchymal stem cells in bovine endometrium during follicular phase of oestrous cycle

Stem cells have been postulated as responsible for cell regeneration in highly and continuously regenerative tissues such as the endometrium. Few studies in cattle have identified and specified the presence of stem cells in the endometrium during the oestrous cycle. The aim of this study was to investigate the presence of mesenchymal stem cells (MSCs) in the bovine endometrium during the follicular phase (FP) of the oestrous cycle. Uterine tissue was collected in the time‐frame comprising day 18 of the cycle and ovulation (day 0). We isolated, cultured and expanded four primary cell lines from endometrium and identified byRT‐qPCR the expression of OCT4, SOX2 but not NANOG (undifferentiated/embryonic markers), CD44 (MSCs marker) and c‐KIT (stem cell marker) genes; and the encoded Oct4, Sox2 and Cd44 proteins by Western blot or immunostaining of paraffin‐embedded tissue in endometrium. We demonstrated that cells isolated from bovine endometrium displayed essentially the same gene expression pattern; however, at the protein level, Oct4 and Cd44 were not detected. Besides, they showed typical functional characteristics of MSCs such as fibroblast‐like morphology, plastic adherence, high proliferative capacity, clone formation in vitro and the ability to differentiate into chondrogenic, osteogenic and adipogenic lineages. We obtained for the first time an extensive characterization of undifferentiated cells populations contained in the bovine endometrium during the FP of the oestrous cycle.     

Oxytocin synthesis and secretion from bovine corpora lutea exposed in vitro to cycloheximide and colchicine

Two experiments were conducted to study the effects of cycloheximide and colchicine on prostaglandin F₂ (PGF₂)-induced secretion and synthesis of oxytocin in bovine luteal tissue in vitro. Corpora lutea were collected from beef heifers on Day 8 of the estrous cycle. In Experiment 1, incorporation of [¹⁴C]-leucine into oxytocin synthesized and secreted by luteal slices after exposure to PGF₂, cycloheximide and cycloheximide plus PGF₂ was examined. In Experiment 2, synthesis and secretion of oxytocin were evaluated in luteal slices incubated with colchicine and PGF₂ alone and in combination. Cycloheximide inhibited incorporation of labeled leucine into luteal proteins by more than 90% and no labeled oxytocin was detected in the media or tissue. Prostaglandin F₂ induced significant secretion of oxytocin that was not inhibited by cycloheximide. Tissue levels of oxytocin after incubation with cycloheximide and/or PGF₂ did not differ and were similar to those of the incubated control. Colchicine alone did not suppress oxytocin secretion and did not alter the ability of PGF₂ to induce significant secretion of this nonapeptide. Tissue concentrations of oxytocin after incubation with colchicine and/or PGF₂ did not differ. These studies indicate that secretion and replenishment of luteal oxytocin in vitro is not contingent upon de novo protein synthesis. Inability of colchicine to suppress oxytocin secretion and synthesis may have been due to the short duration of exposure of luteal tissue to the drug.