A numerical study of aortic flow stability and comparison with in vivo flow measurements.

The development of an engineering transitional turbulence model and its subsequent evaluation and validation for some diseased cardiovascular flows have been suggestive of its likely utility in normal aortas. The existence of experimental data from human aortas, acquired in the early 1970s with catheter-mounted hot film velocimeters, provided the opportunity to compare the performance of the model on such flows. A generic human aorta, derived from magnetic resonance anatomical and velocity images of a young volunteer, was used as the basis for varying both Reynolds number (Re) and Womersley parameter (α) to match four experimental data points from human ascending aortas, comprising two with disturbed flow and two with apparently undisturbed flow. Trials were made with three different levels of inflow turbulence intensity (Tu) to find if a single level could represent the four different cases with 4000 < Re < 10,000 and 17 < α < 26. A necessary boundary condition includes the inflow "turbulence" level, and convincing results were obtained for all four cases with inflow Tu = 1.0%, providing additional confidence in the application of the transitional model in flows in larger arteries. The Reynolds-averaged Navier-Stokes (RANS)-based shear stress transport (SST) transitional model is capable of capturing the correct flow state in the human aorta when low inflow turbulence intensity (1.0%) is specified.

Multidimensional Analysis of the Adult Human Heart in Health and Disease using Hierarchical Phase-Contrast Tomography (HiP-CT).

Cardiovascular diseases (CVDs) are a leading cause of death worldwide. Current clinical imaging modalities provide resolution adequate for diagnosis but are unable to provide detail of structural changes in the heart, across length-scales, necessary for understanding underlying pathophysiology of disease. Hierarchical Phase-Contrast Tomography (HiP-CT), using new (4th) generation synchrotron sources, potentially overcomes this limitation, allowing micron resolution imaging of intact adult organs with unprecedented detail. In this proof of principle study (n=2), we show the utility of HiP-CT to image whole adult human hearts ex-vivo: one 'control' without known cardiac disease and one with multiple known cardiopulmonary pathologies. The resulting multiscale imaging was able to demonstrate exemplars of anatomy in each cardiac segment along with novel findings in the cardiac conduction system, from gross (20 um/voxel) to cellular scale (2.2 um/voxel), non-destructively, thereby bridging the gap between macroscopic and microscopic investigations. We propose that the technique represents a significant step in virtual autopsy methods for studying structural heart disease, facilitating research into abnormalities across scales and age-groups. It opens up possibilities for understanding and treating disease; and provides a cardiac 'blueprint' with potential for in-silico simulation, device design, virtual surgical training, and bioengineered heart in the future.

Canine oocyte maturation in culture: significance of estrogen and EGF receptor gene expression in cumulus cells.

We examined the role of cumulus cells regarding in vitro maturation of canine oocytes, and investigated estrogen and epidermal growth factor (EGF) receptor gene expression and action on nuclear maturation. Canine cumulus-oocyte complexes (COC) were collected from anestrous and diestrous bitches; only COC with vitelline diameter >100 microm were used. In Experiment 1, expression of estrogen receptor (ER) alpha, ERbeta and EGF-receptor (EGF-R) were determined by reverse transcription-polymerase chain reaction (RT-PCR), using mRNA from the oocyte or cumulus cell. Transcripts for the ERbeta and EGF-R were detected in oocytes and cumulus cells, but no message was detected for ERalpha. In Experiment 2, intact COC and the denuded oocytes were cultured in TCM199 medium supplemented with various concentrations of estradiol-17beta (E(2); 0-10 microg/mL) or EGF (0-100 ng/mL) for 72 h; nuclear maturation was then evaluated. In oocytes cultured within intact COC, the rate of germinal vesicle breakdown (GVBD) was higher in the 1 microg/mL E(2) supplemented group (P<0.05), and the rate of metaphase I (MI) was higher in the 10 ng/mL EGF supplemented group, than in the non-supplemented group (P<0.05). However, supplementation of E(2) or EGF to denuded oocytes failed to promote nuclear maturation. In Experiment 3, intact COC were cultured in TCM199 supplemented with 1 microg/mL E(2), 10 ng/mL EGF, and 10% fetal bovine serum (FBS) for 72 h, and nuclear maturation was evaluated. There was no significant difference in the rate of metaphase II (MII) between the medium only, E(2)+EGF, and FBS supplement groups. When E(2) and EGF in combination with FBS were supplemented, the rate of MII was higher than in other groups (P<0.05). We inferred that cumulus cells were involved in mediating the stimulatory effects of E(2) and EGF on nuclear maturation of canine oocytes, and that E(2) and EGF in combination with FBS promoted the completion of oocyte meiotic maturation.

Immunohistochemical mapping of natriuretic peptide receptor-A in the brainstem of Macaca fascicularis.

Natriuretic peptide receptor-A (NPR-A) mediates the biological effects of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP), and is involved in maintaining cardiovascular homeostasis. In this immunohistochemical study we examined the distribution of NPR-A in the brainstem of the cynomolgus monkey. NPR-A immunoreactivity was localized to neurons in specific brainstem regions. NPR-A-immunoreactive perikarya were found in the red nucleus and the oculomotor nucleus in the midbrain, the parabrachial nucleus and the locus coeruleus in the pons, and the dorsal motor nucleus of the vagus, the hypoglossal nucleus, the cuneate nucleus, the gracile nucleus, the nucleus ambiguus, the lateral reticular nucleus, the reticular formation, and the inferior olivary nucleus in the medulla oblongata. Extensive networks of immunoreactive fibers were apparent in the red nucleus, the oculomotor nucleus, the principal sensory trigeminal nucleus, and the parabrachial nucleus. Double immunostaining revealed NPR-A immunoreactivity in cholinergic neurons of the parabrachial nucleus, the dorsal motor nucleus of vagus, the hypoglossal nucleus, and the nucleus ambiguus. However, there was no colocalization of NPR-A and tyrosine hydroxylase in the locus coeruleus. The wide anatomical distribution of NPR-A-immunoreactive structures suggests that natriuretic peptides, besides having a role in the central regulation of endocrine and cardiovascular homeostasis, may also mediate diverse physiological functions.

Reduction of mucin-1 gene expression associated with increased Escherichia coli adherence in the canine uterus in the early stage of dioestrus.

The relation between adherence of Escherichia coli and expression of mucin-1 (Muc1: an integral membrane mucin) mRNA in the endometrium was studied in beagle bitches at different stages of the oestrous cycle and in those with cystic endometrial hyperplasia/pyometra complex (pyometra). The number of E. coli adhering to the endometrium was low at pro-oestrus and oestrus and increased at the early stage (day 10) of dioestrus, corresponding to the implantation period; it declined thereafter. Adhesion of the organisms to endometrial epithelial cells collected at day 10 of dioestrus was inhibited by the addition of D-mannose. When endometrial epithelial cells collected at pro-oestrus were treated with hyaluronidase, an enzyme that digests mucins, the numbers of E. coli adhering to the cells tended to increase. With polymerase chain reaction analysis it was possible to detect Muc1 gene transcripts in the endometrium at all stages of the oestrous cycle, although the level of Muc1 mRNA decreased by day 10 of dioestrus. The levels of Muc1 mRNA in bitches with a clinical stage of pyometra were low and comparable to those at day 10 of dioestrus. The number of E. coli adhering to the endometrium and Muc1 mRNA levels in the endometrium were inversely correlated (r=-0.77, P<0.01). Immunohistochemical analysis showed little staining for Muc1 in the endometrial epithelia at day 10 of dioestrus and in bitches with pyometra. These results suggest that reduction of Muc1 expression is associated with increased E. coli adherence in the canine uterus at the early stage of dioestrus, possibly facilitating the development of pyometra.

Stable maintenance of monkey embryonic stem cells in the absence of bFGF.

Monkey embryonic stem (ES) cells are useful tools in preclinical studies of gene therapy and tissue engineering as well as in primate developmental biology. However, their maintenance is not easy, requiring addition of bFGF to the medium. Herein, we have described a stable, cost-effective method that does not require bFGF. We used a high-density (1 to 1.5x10(5) cells/cm2) of mouse embryonic fibroblasts (MEF) as feeder cells to successfully maintain undifferentiated monkey ES cells for 2 years (approximately 150 passages). Furthermore, these ES cells were competent for electroporation of enhanced green fluorescent protein (EGFP) and subsequent drug selection procedures. We were able to establish EGFP-expressing cell lines using this culture condition. These cell lines expressed undifferentiated markers, such as alkaline phosphatase, SSEA-4, TRA-60, and TRA-81. In addition, strong EGFP expression was observed after differentiation into cardiomyocytes, neurons, or adipocytes, suggesting that these cell lines are a useful tool to study cell transplantation. This method simplifies the culture of monkey ES cells.

Effect of co-culturing with embryonic fibroblasts on IVM, IVF and IVC of canine oocytes.

We studied the effects of mouse embryonic fibroblasts (MEF) and canine embryonic fibroblasts (CEF) on IVM, IVF and IVC of canine oocytes. Cumulus-oocyte complexes were harvested from ovaries by slicing, and in vitro maturation was evaluated in three different conditions: culture media only (control), co-culture with MEF, or co-culture with CEF. The oocytes were cultured for 48 or 72 h. Only oocytes larger than 100 microm in diameter with a homogeneous dark cytoplasm and two or more layers of cumulus cells were used. The culture medium was TCM 199+10% fetal bovine serum (FBS) with 100 IU/mL penicillin and 100 microg/mL streptomycin. After 48 h of IVM, the oocytes were fertilized in vitro with fresh canine spermatozoa that had been selected by a swim-up method, and the oocytes and spermatozoa were co-cultured in modified Krebs-Ringer bicarbonate solution (TYH) for up to 20 h in 5% CO2 in air at 38.5 degrees C. After insemination, oocytes were transferred to three different conditions (the same as for IVM) and were cultured. After 48 or 72 h of maturation in vitro, the maturation rate of MII oocytes cultured in co-culture of MEF and CEF was higher than for oocytes cultured in control (P<0.05). Although the rate that reached the MII stage was not different in the 48 and 72 h cultures, the percentage of degenerated oocytes was greater at 72 h in all three treatment groups. The proportion of monospermic and polyspermic oocytes was not different among the three treatment groups. Cleavage rates were higher in the MEF and CEF treatment groups than in the control group (P<0.05). Co-culture with CEF developed the embryo up to the 16-cell stage, and with MEF up to morula stage. In conclusion, co-culture of embryonic fibroblast cells enhanced nuclear and cytoplasmic maturation of canine oocytes.

Lactoferrin expression in the canine uterus during the estrous cycle and with pyometra.

The expression of lactoferrin, a non-specific antimicrobial defence, in the canine uterus during the normal estrous cycle and in bitches with pyometra was examined. Using polymerase chain reaction analysis, lactoferrin gene transcripts were detected in the endometrium at all stages of the estrous cycle, with the highest levels in estrus. In normal bitches, endometrial lactoferrin mRNA increased from proestrus to estrus (P<0.05). Thereafter, it dramatically decreased from estrus to Day 10 of diestrus (P<0.05), and stayed low at Day 35 of diestrus and anestrus; this was consistent with blood estrogen concentrations. Levels of lactoferrin mRNA were higher in bitches with pyometra than in normal diestrus (P<0.05). With immunohistochemistry, distinct staining of lactoferrin was detected in the luminal and glandular epithelial cells of the endometrium at proestrus and estrus, but little staining was detected at Day 10 of diestrus. At Day 35 of diestrus and anestrus, a partial and weak reaction was present in the same region. In bitches with pyometra, the glandular epithelial cells and many cells in the uterine stroma were strongly stained. Staining cells in the stroma were morphologically similar to neutrophils. No lactoferrin staining was seen in the uterine stromal cells or myometrium in any section. These results suggest that, in the canine uterus, lactoferrin expression is related to the blood concentration of estrogen, and that the dramatic reduction in lactoferrin observed at the early stage of diestrus may impair antimicrobial defense. Also, enhanced expression of lactoferrin mRNA in the endometrium with pyometra may be associated with neutrophil invasion into the uterus to combat the infection.

Cytoplasmic liberation of protein gene product 9.5 during the seasonal regulation of spermatogenesis in the monkey (Macaca fuscata).

Primate spermatogenesis is distinguished by yet unidentified mechanisms to regulate its spermatogenetic activity. In contrast to the well documented hormonal regulators, the cellular events responsible for the regulation of the spermatogenesis has not been addressed. By using PGP 9.5-immunohistochemistry, our previous study demonstrated that the monkey spermatogonia are divided into two distinct sub-populations, i.e. cytoplasmic PGP 9.5-positive and cytoplasmic PGP 9.5-negative spermatogonia. By comparing the cytoplasmic expression of PGP 9.5 between the breeding and nonbreeding seasons of the Japanese monkey (Macaca fuscata) in association with PCNA labeling, the present study demonstrates that the cytoplasmic PGP 9.5-positive Ap spermatogonia significantly increases when the spermatogenetic activity declines in the nonbreeding season. An ultrastructural subcellular localization of PGP 9.5 suggests that the increase of the cytoplasmic PGP 9.5 expression is due to a liberation of PGP 9.5 molecule from the nucleus into the cytoplasm. The results provide a theoretical basis by which PGP 9.5 serves as a novel marker for spermatogonial subtypes, which will have further implications for future studies on spermatogenesis. The analysis using this novel marker suggests that the Ap spermatogonia is a key stage to regulate the amount of the sperm produced in response to the hormonal regulators, and the cytoplasmic liberation of PGP 9.5 may serve as a pivotal phenomenon that enables the fully restorable, transient suppression of spermatogenesis in primate.

Angiotensin II induces the release of 19-hydroxyandrostenedione in man.

Plasma concentrations of 19-hydroxyandrostenedione [19-hydroxy-4-androstene-3,17-dione (19-OH-A-dione)], which we reported to be an amplifier of the action of aldosterone on the basis of results obtained in bioassays using adrenalectomized rats, were measured in man using a specific and sensitive RIA. Plasma 19-OH-A-dione concentrations (mean +/- SE) in normal subjects were 56 +/- 4 pg/ml in men and 51 +/- 4 pg/ml in women. Plasma 19-OH-A-dione rose significantly during ACTH stimulation and declined significantly during dexamethasone suppression. Plasma 19-OH-A-dione levels in the adrenal vein were an order of magnitude higher than those in the inferior vena cava. These results demonstrate that 19-OH-A-dione is directly secreted by the adrenal cortex and that its secretion is under the control of ACTH. As the action of 19-OH-A-dione may be closely related to that of aldosterone, the response of 19-OH-A-dione to angiotensin II infusion was evaluated and compared with that of aldosterone. During the infusion of graded doses (0.5-4.0 ng/kg . min) of angiotensin II, plasma 19-OH-A-dione increased significantly after the infusion of angiotension II at rates of 1.0 and 2.0 ng/kg . min. It then declined after the infusion of angiotensin II at a rate of 4.0 ng/kg . min. In contrast, plasma aldosterone did not increase significantly until the infusion rate reached 4.0 ng/kg . min. These results indicate that the secretion of 19-OH-A-dione is under the control of angiotensin II. Similar changes in 19-OH-A-dione and aldosterone were found during postural changes. In those subjects who had a small increase in plasma aldosterone when they were upright, 19-OH-A-dione significantly increased. In contrast, in those subjects who had a large increase in plasma aldosterone when upright, 19-OH-A-dione significantly declined. These results suggest that a small elevation of endogenous angiotensin II induces an elevation of plasma 19-OH-A-dione, and a sharp increase in endogenous angiotensin II induces an increase in plasma aldosterone and a decline in plasma 19-OH-A-dione. As angiotensin II stimulated the release of 19-OH-A-dione when the secretion of ACTH was suppressed, angiotensin II acted directly on the adrenal cortex. 19-OH-A-dione is a newly recognized biologically active adrenal C19 steroid which is regulated by both ACTH and the renin-angiotensin system in man.

Angioscopic evaluation of the effect of estrogen on ovariectomized Japanese monkeys.

Using angioscopy to study the atheroprotective effect of estrogen, atherosclerotic changes in surgically postmenopausal Japanese monkeys were observed for a long period. Changes in serum lipids and estradiol were also followed. About 3-6 months after the 2% cholesterol diet, serum cholesterol increased up to three times normal value, and atheroma was first found at the orifice of major branches of the abdominal aorta. However, in the other group of 2% cholesterol including an oral estrogen diet, the appearance of atheroma was inhibited until 12 months, although serum cholesterol increased gradually. The chronologic investigation was continued over 3 years. From these results we concluded that the most important atheroprotective effect of estrogen is the direct inhibition of atherosclerosis in the arterial wall, and improvement in lipid metabolism is of secondary importance.

New hyperprolactinemia and anovulation model in common marmoset (Callithrix jacchus) and effect of cabergoline.

We aimed to develop an anovulation model, using sulpiride-induced hyperprolactinemia in common marmosets. The serum prolactin level gradually increased during the twice-daily administration of sulpiride and reached a plateau after 4 days. Sulpiride produced as big a response at 10 mg kg(-1) as at 50 mg kg(-1). In this study, the length of the ovarian cycle was approximately 30 days in normal common marmosets. Serum progesterone and estradiol levels showed no consistent change during the first 2 months of treatment with sulpiride. When treatment with sulpiride had been continued for more than 2 months, serum progesterone and estradiol levels fell to within the range seen in the follicular phase of the normal cycle and absence of ovulation was recognized by laparoscopy. A single oral administration of cabergoline (at doses between 0.01 and 0.1 mg kg(-1)) dose dependently reduced the elevated serum prolactin level. Bromocriptine (at an oral dose of 10 mg kg(-1)) also reduced the serum prolactin level at 4 and 8 h after its administration. With bromocriptine, the prolactin level had recovered at 24 h, but with cabergoline at doses of 0.05 mg kg(-1) or more, it had still not recovered at 48 h. In anovulatory common marmosets, oral administration of cabergoline at a daily dose of 0.05 mg kg(-1) restored ovarian function and resulted in ovulation in 100% of the group (following a reduction in the serum prolactin level). Bromocriptine at a daily oral dose of 10 mg kg(-1) resulted in ovulation in 67% of the group, but this dose was about 200 times higher than the dose of cabergoline. We could produce an anovulatory model induced by sulpiride repeatedly administered over a long time period. It is suggested that, in this anovulatory model in common marmosets, cabergoline has a potent and long-lasting action as a dopamine D2 receptor agonist, and thus could be a useful drug for the treatment of galactorrhea and hyperprolactinemic amenorrhea and/or anovulation.

Enhancement of estrogen receptor gene expression in the mediobasal hypothalamus during anestrus in the beagle bitch.

Estrogen receptor mRNA (ER mRNA) levels were measured in the mediobasal hypothalamus (MBH) of beagle bitches at different stages of the estrous cycle, and compared with levels in ovariectomized (OVX) estrogen-treated bitches. In cyclic bitches, the level of hypothalamic ER mRNA increased during the progression of anestrus and declined thereafter. Hypothalamic ER mRNA and plasma luteinizing hormone (LH) levels during anestrus and proestrus were positively correlated (r = 0.94, P < 0.001). In OVX bitches, levels of hypothalamic ER mRNA were low, and increased significantly after treatment with a low dose of estradiol benzoate. These results suggest that, during the course of anestrus in the bitch, hypothalamic ER mRNA expression increases, and may be up-regulated by estradiol.

Increasing gonadotropin-releasing hormone release by perifused hypothalamus from early to late anestrus in the beagle bitch.

An in vitro perifusion system was used to investigate pulsatile gonadotropin-releasing hormone (GnRH) release from hypothalamic fragments derived from beagle bitches at different stages of the estrous cycle. The spontaneous GnRH release from the excised tissue fragments that include the "mediobasal hypothalamic-preoptic area-suprachiasmatic nucleus units' was episodic throughout all stages of the estrous cycle with a significantly high release rate during late anestrus and late proestrus. The GnRH release rate and plasma levels of luteinizing hormone were positively correlated (r = 0.94, P < 0.01). These results suggest that during the course of anestrus in the bitch the GnRH release rate increases while the pituitary responds accordingly.

Hyperlipidemia and atherosclerosis in experimental insulinopenic diabetic monkeys.

Chronic insulinopenic diabetes was induced by i.v. streptozotocin in the non-human primate Macaca fuscata. Five diabetic monkeys were kept for 8-19 months and nine for 24-48 months without any insulin treatment. Hyperglycemia (241 +/- 22 mg/dl, M +/- SE less than or equal to 1 year) progressed to 376 +/- 34 mg/dl (greater than 2 years) and ketosis to 3.5 mM (greater than 2 years) during the course of diabetes; this was roughly inversely proportional to hypoinsulinemia (3.4 microU/ml, 2 years). Serum cholesterol increased from 184 +/- 11 (less than or equal to 1 year) to 328 +/- 66 mg/dl (greater than 2 years) with the major increase in LDL-cholesterol (2.7-fold over control, greater than 2 years). HDL-cholesterol did not change at all throughout the experimental period. TG increased from 144 +/- 25 (less than or equal to 1 year) to 676 +/- 116 (greater than 2 years) with a major increase in the VLDL fraction (15-fold over control, greater than 2 years). Serum levels of apo B increased to 141 +/- 16 (less than or equal to 2 years) and 223 +/- 8 mg/dl (greater than 2 years) in contrast to control, 73 +/- 2. Morphologically, lipid deposition in the intima and fatty streaks have been observed in the abdominal aorta of all the diabetic monkeys with duration of more than 2 years. In six of the diabetic monkeys atheromatous changes such as intimal and medial thickening with smooth muscle cell proliferation were observed with foam cell formation. Similar atherosclerotic lesions were observed in renal and coronary arteries in at least six of these monkeys. In diabetic monkeys with duration of less than 2 years, mild atherosclerotic lesions were observed in two out of five. The results indicate that long standing insulinopenia leads to metabolic derangements characterized by hyperglycemia, ketonemia and hyperlipidemia. Elevation of LDL-cholesterol and VLDL TG with an increase of apo B is a characteristic of lipoprotein disorder. Morphologically, early to moderately advanced lesions of atherosclerosis were observed in aorta, renal and coronary arteries as a result of metabolic derangement due to insulin deficiency.

Hypovolemic intolerance to lower body negative pressure in female runners.

PURPOSE: An attenuated baroreflex response and orthostatic intolerance have been reported in endurance-trained male athletes; however, it is still unknown whether this occurs also in females. The purpose of the present study was to examine whether endurance exercise-trained women had a predisposition to orthostatic compromise, and if so, what causative factor(s) may induce orthostatic intolerance. METHODS: We studied cardiovascular and hormonal responses to graded lower body negative pressure (LBNP) (0 to -60 mm Hg) in 26 middle-distance female runners (18.6 +/- 0.1 yr) as the exercise-trained (ET) subjects and 23 age-matched untrained (UT) control subjects. On the basis of the occurrence of syncope episodes during LBNP, ET and UT subjects were further allocated to two groups; ET with presyncope (ET+syncope) and without presyncope (ET-syncope) and UT with presyncope (UT+syncope) and without presyncope (UT-syncope). RESULTS: Occurrence of presyncope episodes during LBNP was higher in ET (65.4%, P < 0.05) than that for UT (34.8%). Leg compliance was higher (P < 0.05) in ET than in UT. LBNP reduced stroke volume (SV) more (P < 0.05), increased heart rate (HR) higher (P < 0.05), and increased forearm vascular resistance (FVR) more in ET+syncope as compared with the other groups. Response of vasoactive hormones to LBNP was higher in ET+syncope (P < 0.05) than that of the other groups except for norepinephrine (NE); high in both ET+syncope and UT+syncope. The relationship between SV and NE, an index of sympathetic neuronal response, had no training-related changes during LBNP. CONCLUSION: We conclude that exercise-trained females have a high incidence of orthostatic intolerance during LBNP, with a greater reduction of SV independent of changes in baroreflex and neurohumoral function. A lower incidence of LBNP intolerance in UT may be accounted for by a lower reduction of SV during LBNP. An increase in leg compliance in the exercise-trained females may play an important role in inducing pronounced reduction of SV and hence the intolerance to LBNP.

Reversible suppression of pituitary-testicular function by a sustained-release formulation of a GnRH agonist (leuprolide acetate) in dogs.

Pituitary-testicular function was studied in 15 dogs following treatment with a sustained-release formulation of a GnRH agonist, leuprolide acetate (LA). Adult male dogs were treated with a single subcutaneous injection of microencapsulated LA (0.1 or 1 mg/kg). Treatment with LA at a dose of 1 mg/kg resulted in decreased (P<0.001) ejaculatory volume and disappearance of morphologically normal spermatozoa within 8 wk and the effect persisted for 6 wk, while the 0.1 mg/kg dose was not adequate to effect suppression of spermatogenesis. The larger dose treatment (1 mg/kg) caused a transient rise in plasma levels of LH and testosterone followed by a marked decline to below the normal level by 2 wk, the low levels being maintained for at least 5 wk, indicating a prolonged effect of LA treatment on pituitary-gonadal axis. Twenty weeks after treatment with LA, a complete return to normal spermatogenesis was observed. The full reversibility of spermatogenesis in the dog after LA treatment suggests that this peptide could be used as a reversible method of male contraception.

Induction of fertile estrus in bitches using a sustained-release formulation of a GnRH agonist (leuprolide acetate).

A single subcutaneous injection of a sustained-release formulation of a potent GnRH agonist, leuprolide acetate (LA; [D-Leu6, Pro9NEt]-GnRH), was evaluated as a method of inducing fertile estrus in 12 mature anestrous and 6 prepubertal beagle bitches. The bitches were treated with microencapsulated LA (100 micrograms/kg, s.c.) at 120 or 150 d post partum, or at 1 yr of age, followed by a GnRH-analogue (fertirelin; [Pro9NEt]-GnRH, 3 micrograms/kg, i.m.) on the first day of induced estrus. Signs of estrus were seen within 10.3 +/- 0.9 d after LA administration in all bitches. The interestrous interval in 120- and 150-d post-partum bitches was shortened (P < 0.05) to 191 +/- 3 and 222 +/- 3 d, respectively, compared with 264 +/- 11 d in control bitches. All LA treated dogs demonstrated behavioral estrus and mated. Three of 6 (50%) at 120 d post partum, 6 of 6 (100%) at 150 d post partum and 5 of 6 (83%) of prepubertal (1-yr old) bitches then became pregnant and produced a mean litter size of 4.1 +/- 0.8 pups. A normal circulating estrogen and progesterone response pattern was observed in mature anestrous bitches. A prepubertal bitch that failed to become pregnant had a similar estrogen response pattern but an insufficient progesterone profile. The results suggest that microencapsulated LA can be useful in inducing fertile estrus in the domestic dogs.

Increased LH pulse frequency and estrogen secretion associated with termination of anestrus followed by enhancement of uterine estrogen receptor gene expression in the beagle bitch.

The relationships among pulsatile LH secretion pattern, estrogen secretion, and expression of the uterine estrogen receptor gene were examined throughout the estrous cycle in beagle bitches. In Experiment 1, blood samples were collected from 30 bitches every 10 min for 8 h from a cephalic vein during different phases of the estrous cycle. An increase in the mean plasma levels of LH occurred from mid to late anestrus (P < 0.01). The LH pulse frequency increased (P < 0.01) from late anestrus to proestrus, and was strongly correlated (r = 0.96, P < 0.001) with the mean plasma level of estradiol-17 beta (E2). In Experiment 2, middle uterine samples, including the myometrium and endometrium, from 18 bitches were taken at 6 stages of the estrous cycle. The total number of estrogen receptors and nuclear estrogen receptor and its mRNA levels in the uterus also increased (P < 0.01) from late anestrus to proestrus. Mean plasma E2 level and the number of uterine estrogen receptor were positively correlated (r = 0.81, P < 0.05). In Experiment 3, nine bitches were ovariectomized in mid anestrus. Two weeks later they received a single injection of 10 or 50 micrograms/kg, i.m., estradiol benzoate. The number of uterine estrogen receptor and their mRNA levels for ovariectomized bitches were low, but increased (P < 0.05) after treatment with a low dose of estradiol benzoate. These results suggest that increases in LH pulse frequency and estrogen secretion are associated with termination of anestrus and that subsequent enhancement of uterine estrogen receptor expression may be up-regulated by estradiol.

Developmental changes in testicular luteinising hormone receptors and androgens in the dog.

The changes with age in the levels of testicular luteinising hormone (LH) receptors in dogs, were correlated with plasma androgen concentrations and the effects of stimulation with human chorionic gonadotrophin (hCG). The presence of high-affinity binding sites for LH was demonstrated in the testes of all the animals studied, and the apparent affinity of the LH receptors did not change significantly with age. The total number of LH receptor sites per testis increased continuously with age and the growth of the testis. The major rise in the number of LH receptors occurred between six and 10 months of age, and coincided with the phase of rapid testicular growth. The concentrations of androstenedione, testosterone and 5 alpha-dihydrotesterone in plasma increased gradually until six months of age, and then increased rapidly with the increase in the number of testicular LH receptors. An increase in the concentrations of androgens in plasma was observed in response to stimulation with hCG after the number of testicular LH receptors began to increase progressively. The increase in the sensitivity of the dogs' gonads to LH could be responsible for the marked increase in the secretion of androgens which occurs during puberty in the male dog.