Conceptus-mediated endometrial vascular changes during early pregnancy in mares: an anatomic, histomorphometric, and vascular endothelial growth factor receptor system immunolocalization and gene expression study.

This work examined how the conceptus modulates endometrial tissue remodeling and vascular development prior to implantation in mares. A macroscopic uterine examination was completed at day 21 of pregnancy. In situ morphology revealed that the endometrium involved in encroachment is restricted to the dorsal endometrium immediately overlying the yolk sac. The amount of stromal area occupied by blood vessels and the number of endometrial glands were increased during early pregnancy. Endometrial histomorphometry as well as the endometrial mRNA abundance and immunolocalization of VEGF, VEGFR1, VEGFR2, and Ki-67 was completed at days 14 and 21 of pregnancy, at day 10 of the estrous cycle, and during estrus. No obvious differences in VEGF and VEGFR1 protein localization were detected between pregnant and cycling mares but differential staining pattern for VEGFR2 and Ki-67 was observed. VEGFR2 localized to luminal and glandular epithelium of pregnant mares, while luminal epithelium was negative in cycling mares. Ki-67 staining was weak during the luteal phase but exhibited prominent luminal epithelium staining during estrus. In pregnant mares, all endometrial layers were Ki-67 positive. Quantitative RT-PCR revealed a greater abundance of VEGF mRNA during pregnancy. VEGFR2 transcript abundance was greatest in pregnant mares on day 21. This study supports the concept that the conceptus plays an active role in directing vasculogenesis within the uterus and thereby establishing hemotrophic nutrition that supports pregnancy after implantation.

A single gene encodes the beta-subunits of equine luteinizing hormone and chorionic gonadotropin.

Equine (e) CG and LH beta-subunits have identical amino acid sequences, including an extended carboxyl-terminal peptide (CTP). This suggests that unlike the corresponding human genes, the beta-subunits of eCG and eLH may be encoded by a single gene and share a common proximal promotor region. To explore this, we isolated and characterized the eLH/CG beta gene(s). Data from Southern analyses suggest that the eCG beta and eLH beta subunits are products of the same single copy gene (eLH/CG beta). Overlapping fragments of the eLH/CG beta gene and cDNA were amplified from equine genomic DNA and pituitary gland mRNA by the polymerase chain reaction, cloned, and sequenced. The eLH/CG beta gene spans less than 1.2 kilobase-pairs and has three exons that translate a CTP-containing polypeptide identical in sequence to that previously reported for the mature equine protein. There is, however, little amino acid homology shown between the CTP of human or equine CG beta subunit. In addition, unlike the human genes, the same TATAA-like element appears to be involved in directing initiation of transcription of the eLH/CG beta gene in placenta and anterior pituitary. Based upon these differences, we suggest that the CG beta genes evolved independently in humans and equids and that different mechanisms are involved in their patterns of placenta-specific expression.

Endocrine aspects of early pregnancy in pony mares: a comparison of uterine luminal and peripheral plasma levels of steroids during the estrous cycle and early pregnancy.

Comparisons of estrone, 17 beta-estradiol, and plasma progestin concentrations were made in uterine fluid and peripheral blood of nonpregnant and pregnant pony mares. Concentrations of these steroids were also measured within yolk sac fluid from blastocysts on days 12, 14, 16, and 18 of pregnancy to obtain more complete analyses of the uterine environment (uterine fluid plus yolk sac fluid) of early pregnancy. Thirty mares were randomly assigned to six treatment groups (n = 5/group), and uterine fluid and peripheral blood samples were obtained on days 8, 12, 14, 16, 18, and 20 postovulation. After a recovery period of one estrous cycle, mares were bred at their next estrus. Animals were hysterectomized on the same treatment day to which they had previously been assigned in the nonpregnant phase of this study. Using this design, uterine fluid and peripheral blood samples were collected from each mare on equivalent days of the estrous cycle and pregnancy. Significant differences in day trends were found between nonpregnant and pregnant animals for estrogens and progestins in both uterine fluid and peripheral plasma. Furthermore, these data demonstrate that large increases in estrogens occur after day 12 of pregnancy in uterine and yolk sac fluids, with estrone becoming the predominant estrogen by days 18 and 20 in yolk sac and uterine fluids, respectively. These changes were not detected in peripheral plasma, which indicates that changes occurring within the uterine environment are not discernible in the systemic circulation during early pregnancy. These results indicate that the large amounts of estrogens appearing in uterine fluids during early pregnancy are of conceptus origin and may be an important factor in regulating the environment in which the conceptus develops.

Sympathetic ophthalmia. Histopathologic and fluorescein angiographic correlation.

Sympathetic ophthalmia was found 15 months after cataract extraction in a previously traumatized eye. Histopathologic examination disclosed numerous Dalen-Fuchs nodules with underlying choroidal granulomas causing obliteration of the choriocapillaris and disruption of Bruch's membrane. Prominent Dalen-Fuchs nodules were also observed clinically in the sympathizing eye, and the angiographic findings of initial hypofluorescence followed by late hyperfluorescence correlated with the pathologic findings in the sympathogenic eye. Only ten cases of sympathetic ophthalmia with fluorescein angiography have been reported previously. This is the fifth case accompanied by a histopathologic description of the exciting eye. The finding of breaks in Bruch's membrane has not been previously reported, to the best of our knowledge. This article compares the angiographic and pathologic appearances with those described in previous reports.

Real-time monitoring of nitrile biotransformations by mid-infrared spectroscopy.

In this study mid-infrared spectroscopy was used to follow the enzyme kinetics involved in nitrile biocatalysis using whole cell suspensions of the bacterium Rhodococcus rhodochrous LL100-21. The bacteria were grown on acetonitrile to induce a two-step enzymatic pathway. Acetonitrile was biotransformed to acetamide by a nitrile hydratase enzyme and subsequently to acetic acid (carboxylate ion) by an amidase enzyme. The bacteria were also grown on benzonitrile to induce a one-step enzymatic pathway. Benzonitrile was biotransformed directly to benzoic acid (carboxylate ion) by a nitrilase enzyme. These reactions were followed by React IR using a silicon probe and gave excellent quantitative and qualitative real-time data of both nitrile biocatalytic reactions. This study has shown that this novel technique has potentially useful applications in biocatalysis.

The observation of mitotic division aberrations in mammalian cells exposed to chemical and radiation treatments.

The fidelity of chromosome segregation and the maintenance of the integrity of the chromosome karyotype of eukaryotic cells is dependent upon the synthesis and functioning of division-related structures such as the nuclear spindle and events such as the attachment of chromosomes to the spindle and their subsequent movement to the poles of the dividing cell. Chemical and physical treatment which modify the synthesis and functioning of division-related events may potentially lead to the production of cells with abnormal chromosome numbers (of both whole chromosome sets and of individual chromosomes). The ability of environmental agents to modify division-related structures in mammalian cells has been assessed by morphological examination of exposed mitotically dividing cells using staining techniques which identify spindle and chromosome structure and by the analysis of the characteristics of microtubule polymerisation in vitro. Such techniques have been used to identify the spindle-modifying effects of chemicals such as the synthetic hormone diethylstilboestrol and modifications of chromosome to spindle attachment in cells exposed to both UV- and X-irradiation. Such modifications of cell-division-related activities may lead to alterations in the fidelity of division events leading to the production of chromosomally abnormal daughter cells with aneuploid or polyploid karyotypes.

Influence and correction of temperature perturbations on NIR spectra during the monitoring of a polymorph conversion process prior to self-modelling mixture analysis.

The influence of temperature variations on the rank of a NIR dataset, has been investigated by comparing the results of principal component analysis (PCA) and evolving factor analysis (EFA), applied to two datasets measured at constant temperature and varying temperature. After temperature correction, the concentration profiles and spectra were obtained with PCA, SIMPLISMA and the orthogonal projection approach (OPA). The same resolution methods were used on the dataset measured at constant temperature.

The effect of pulsatile gonadotropin-releasing hormone and estradiol administration on luteinizing hormone and follicle-stimulating hormone concentrations in pituitary stalk-sectioned ovariectomized pony mares.

Hourly pulses of gonadotropin-releasing hormone (GnRH) or bi-daily injections of estradiol (E2) can increase luteinizing hormone (LH) secretion in ovariectomized, anestrous pony mares. However, the site (pituitary versus hypothalamus) of positive feedback of estradiol on gonadotropin secretion has not been described in mares. Thus, one of our objectives involved investigating the feedback of estradiol on the pituitary. The second objective consisted of determining if hourly pulses of GnRH could re-establish physiological LH and FSH concentrations after pituitary stalk-section (PSS), and the third objective was to describe the declining time trends of LH and FSH secretion after PSS. During summer months, ovariectomized pony mares were divided into three groups: Group 1 (control, n = 2), Group 2 (pulsatile GnRH (25 micrograms/hr), n = 3), and Group 3 (estradiol (5 mg/12 hr), n = 3). All mares were stalk-sectioned and treatment begun immediately after stalk-section. Blood samples were collected every 30 min for 8 h on the day before surgery (D0) and 5 d post surgery (D5) to facilitate the comparison of gonadotropin levels before and after pituitary stalk-section. Additionally, jugular blood samples were collected every 12 hr beginning the evening of surgery, allowing for evaluation of the gonadotropin secretory time trends over the 10 d of treatment. On Day 10, animals were euthanized to confirm pituitary stalk-section and to submit tissue for messenger RNA analysis (parallel study). Plasma samples were assayed for LH and FSH by RIA. Mean LH secretion decreased from Day 0 to Day 5 in Groups 1 and 3, whereas LH secretion tended (P < 0.08) to decrease in Group 2 mares. On Day 5, LH was higher (P < 0.01) in Group 2 (17.26 +/- 3.68 ng/ml: LSMEANS = SEM), than either Group 1 (2.65 +/- 4.64 ng/ml) or Group 3 (4.28 +/- 3.68 ng/ml). Group 1 did not differ from Group 3 on Day 5 (P < 0.40). Similarly, mean FSH levels decreased in all groups after surgery, yet Group 2 mares had significantly (P < 0.001) higher FSH concentrations (17.66 +/- 1.53 ng/ml) than Group 1 or Group 3 (8.34 +/- 1.84 and 7.69 +/- 1.63 ng/ml, respectively). Regression analysis of bi-daily LH and FSH levels indicated that the time trends were not parallel. These findings indicate: 1) Pituitary stalk-section lowered LH and FSH to undetectable levels within 5 d after surgery. 2) pulsatile administration of GnRH (25 micrograms/hr) maintained LH and FSH secretion, although concentrations tended to be lower than on Day 0, and 3) E2 did not stimulate LH or FSH secretion.

The early fetal life of the equine conceptus.

This paper will discuss development of the equine conceptus, especially from the perspective of the maternal environment in which it develops and to which it has considerable influence.

An equine fetal monster dicephalus tetrapus dibrachius.

Double monsters are structurally related to monozygotic twins and the reported frequency of these abnormalities in the equine species is low. Symmetrical, double development in both the cephalic region and caudal trunk was demonstrated along with a common undeveloped cervical mass. Anomalies found in the separate genito-urinary systems added another variant to this case.

Effects of melatonin implants in pony mares. 2. Long-term effects.

The effects of melatonin implant treatment over a 4 wk period at the summer solstice on the transition into and out of the following anovulatory season were evaluated in ovary-intact and ovariectomized mares. Melatonin implants tended to delay the timing of the final ovulation of the breeding season (P = 0.0797) in the ovary-intact mares. Although the decline in LH secretion associated with the end of the breeding season was parallel between treatments and ovarian statuses, the rate of LH secretion, as expressed by its mathematical accumulation, was lower in ovariectomized, melatonin-treated mares than in ovariectomized, control mares suggesting that melatonin administration advanced the offset of the breeding season in ovariectomized mares (P = 0.0001). The first ovulation of the subsequent breeding season was significantly delayed in the melatonin-treated mares as compared with that of control mares (P = 0.0031). During reproductive recrudescence, the time of the onset of the increase in LH secretion was similar among all 4 groups but the patterns of LH secretion were different for each treatment and ovarian status combination (P = 0.0112). Mares with melatonin implants had a slower rate of increase in LH secretion than control mares (P = 0.0001), and ovariectomized mares had a faster rate of LH increase than intact mares (P = 0.0001). These results suggest that melatonin implants during the summer solstice can alter the annual reproductive rhythm in mares and support the concept that endocrine patterns of reproductive recrudescence are not entirely independent of the ovary.

Effects of melatonin implants in pony mares. 1. Acute effects.

The effects of melatonin implant treatment over a four week period on LH, estradiol (E2) and progesterone (P4) secretion during the breeding season were studied in ovary-intact and ovariectomized pony mares. Mares with melatonin implants had significantly higher daytime melatonin concentrations than mares with sharm implants (P = 0.0065). In ovariectomized mares, LH secretion did not differ between mares with melatonin and sham implants. In ovary-intact mares, melatonin implants altered the pattern of LH secretion (P = 0.0023) in such a way that an increase in LH secretion was observed during the periovulatory period. Estradiol and P4 secretion were unaffected by melatonin implants. These results suggest that constant administration of melatonin may enhance the secretion of LH during the periovulatory surge but does not adversely affect E2, P4 or basal LH secretion in mares during the breeding season.

Effects of estradiol-17beta administration on steady-state messenger ribonucleic acid (MRNA) encoding equine alpha and LH/CGbeta subunits in pituitaries of ovariectomized pony mares.

The process of sexual recrudescence in the springtime in mares is characterized by renewal of follicular growth and acquisition of steroidogenic competence. Concomitant with renewal of follicular steroidogenesis is re-establishment of LH biosynthesis and secretion. Research results from our laboratory indicate that increased estradiol and LH secretion occur in close temporal association before the first ovulation of the year. Therefore, the hypothesis tested in this experiment was that estrogen administration to ovariectomized pony mares during the equivalent time of early vernal transition would enhance LH biosynthesis as monitored by messenger ribonucleic acid (mRNA) encoding for the pituitary subunits of LH (alpha and LH/CGbeta). Mares were administered either sesame oil vehicle control, or estradiol (5 mg i.m. twice daily in sesame oil) for 3, 6 or 9 days, beginning on February 2. The pituitary glands were harvested, and examined for LH subunit mRNA by Northern Blot and slot blot analysis. There was a significant increase in LH secretion after 6 days of estradiol secretion compared with control vehicle administration. Similarly, there was a significant increase in both alpha and LH/CGbeta subunit mRNA when estradiol was administered for 9 days. These data indicate that estrogen stimulates LH subunit formation in mares during early equivalent vernal transition. These data do not, however, discriminate between a direct pituitary effect of estrogen, and a hypothalamic effect. Whether the surge of estradiol just prior to the first ovulation of the year is essential for the renewed biosynthesis of LH subunits cannot be determined from these data. However an important role of estrogen in the final stages of sexual recrudescence is indicated.

Effects of ovarian input on GnRH and LH secretion immediately postovulation in pony mares.

The potential involvement of ovarian factors in regulating GnRH and LH postovulation was studied in ovarian intact (Group 1; n=3) and ovariectomized (OVX; Group 2; n=3) mares (OVX within 12 hr of ovulation). Blood samples were collected every 10 min for 6 hr from jugular vein (JV) and intercavernous sinus (ICS) during estrus and on Day 8 postovulation for LH and GnRH analysis. Additionally, JV samples were collected twice daily (12-hr intervals) for 30 days for LH and progesterone (P4) analysis. A significant treatment x day effect (P<0.0001) describes declining plasma LH concentrations in intact mares, and regression analysis indicated that response curves were not parallel (P<0.001). Plasma LH concentrations remained elevated in OVX mares. LH increased further in OVX mares by Day 8 post-OVX (P<0.06), reflecting the increased (P<0.07) LH episode amplitude. GnRH decreased from estrus to Day 8 in both groups reflecting an effect of sampling period (P<0.03). GnRH episode amplitude declined (P<0.08) from estrus (62.8+/-3.1 pg/mL) to Day 8 (46.3+/-3.1 pg/mL) in OVX mares, but not in control mares (intact estrus, 36.5+/-6.4; intact Day 8, 37.5+/-7.3; OVX estrus, 62.8+/-3.1; OVX Day 8, 46.3+/-3.1 pg/mL). In conclusion, we propose that postovulatory LH decline requires ovarian feedback in mares, and that OVX alters GnRH secretory dynamics such that LH concentrations does not decline postovulation and, in fact, is further elevated with time after OVX.

Effect of beta-carotene administration on reproductive function of horse and pony mares.

The objective of this study was to determine whether supplemental beta-carotene would influence reproductive function in mares maintained on spring and summer pastures and to characterize plasma carotene concentrations during the estrous cycle. Carotene concentrations in plasma did not vary with day of estrous cycle (P = 0.7455). Mares receiving every other day injections of beta-carotene (400 mg; n = 4) or saline (10 ml; n = 4) during proestrus/estrus did not differ in plasma estradiol (E(2)) concentrations (P = 0.6313), follicle development (P = 0.8068), or plasma progesterone (P(4)) concentrations during the following diestrus (P = 0.4954). Moreover, no differences in plasma P(4) concentrations (P = 0.9047) were detected between mares receiving every other day injections of beta-carotene (400 mg; n = 4) or saline (10 ml; n = 4) during diestrus. However, administration of beta-carotene raised plasma carotene concentrations relative to controls when injected during proestrus/estrus (P = 0.0096) and diestrus (P = 0.0099). Pregnancy rates (P = 0.4900) and number of cycles required for pregnancy (P = 0.2880) were similar for mares administered injections of saline (10 ml; n = 37), beta-carotene (400 mg; n = 37), vitamin A (160,000 IU; n = 38), or vitamin A + beta-carotene (160,000 IU + 400 mg; n = 43), on the first or second day of estrus and on the day of breeding. Therefore, these results collectively suggest that supplemental beta-carotene does not affect the reproductive function of mares fed adequate dietary carotene. Whether supplemental beta-carotene would enhance reproductive function in mares on low carotene diets warrants further investigation.

Technical note: Application of the Box-Cox data transformation to animal science experiments.

In the use of ANOVA for hypothesis testing in animal science experiments, the assumption of homogeneity of errors often is violated because of scale effects and the nature of the measurements. We demonstrate a method for transforming data so that the assumptions of ANOVA are met (or violated to a lesser degree) and apply it in analysis of data from a physiology experiment. Our study examined whether melatonin implantation would affect progesterone secretion in cycling pony mares. Overall treatment variances were greater in the melatonin-treated group, and several common transformation procedures failed. Application of the Box-Cox transformation algorithm reduced the heterogeneity of error and permitted the assumption of equal variance to be met.

Interrelationships between uterus and conceptus to maintain corpus luteum function in early pregnancy: sheep, cattle, pigs and horses.

Processes associated with "Maternal Recognition of Pregnancy" are reviewed extensively from the ovine, bovine, porcine and equine species. Comparisons among these species indicate that CL maintenance is achieved primarily by a predominant antiluteolytic-anti PGF effect, and there is strong evidence for antiluteolytic-luteoprotective and luteotropic controls that complement this basic system. The nature of the chemical signals (steroids, prostaglandins and proteins) to regulate these processes among the species are described. Common to all of the species reviewed is a change in vascular dynamics to and from the uterus and ovary during early pregnancy. The dialogue between endometrial epithelium and trophectoderm of the developing conceptus is described. The consequence of these various physiological and biochemical responses of early pregnancy is maintenance of the CL to provide a sustained embryotrophic environment. Either in the absence of or death of a conceptus, an efficient and acute system is operational to terminate this progestational environment via regression of the CL through uterine production of PGF.

Anterior segment necrosis following cyclocryotherapy.

A 74-year-old woman with secondary glaucoma underwent cyclocryotherapy. Ten weeks later anterior segment necrosis and dehiscence in the area of the cryotherapy lesions resulted in an expulsive choroidal hemorrhage. Anterior segment ischemia following cyclocryotherapy has previously been described, but this is the first reported case of anterior segment necrosis as a complication of this procedure.

Luteinizing hormone during sexual maturation in pony mares.

Luteinizing hormone (LH) was quantified in pony mares during artificially induced sexual maturation. Ovarian follicular development was also assessed by rectal palpation of the ovaries. With the exception of large periovulatory LH concentrations in two mares that ovulated, LH concentrations were not significantly different in mares undergoing sexual maturation and thus having marked follicular development when compared with mares that were not undergoing sexual maturation and thus did not have marked follicular development. These results indicate a dissociation in time between the onset of follicular development and the increase in LH which is associated with the first ovulation of the year.

A technique for chronic, extraluminal measurement of uterine activity.

The construction, calibration, and surgical placement of a device for monitoring uterine motility are described. The device, a linear stretch gauge, consisted of a length of flexible tubing filled with mercury and connected at both ends to copper wire leads. An increase in the length of the mercury-filled tubing caused a change in resistance. This change was quantitated, using a modified Wheatstone bridge circuit. In in vitro test, the stretch gauges demonstrated linear response in millivolt output to elongation over a range that was comparable to physiologic responses observed with the gauges placed in vivo. If surgically placed around 1 uterine horn, stretch gauges responded to uterine contractile events with specificity and sensitivity. Calibration of the device at time of placement permitted monitoring for possible increases in uterine circumference.