Sub-chronic exposure to dibromoacetic acid, a water disinfection by-product, does not affect gametogenic potential in mice.

Water disinfection by-products, such as dibromoacetic acid (DBA), are formed when drinking water is treated with chlorination, bromination, or ozonation. Epidemiological studies have linked these byproducts to adverse effects in humans such as cancer, developmental defects, and reproductive toxicities. DBA has been shown to produce reproductive toxicity in rodents at relatively high doses. The present study used a mouse model to determine the developmental and reproductive effects of sub-chronic, low-dose exposure to DBA. Pregnant mice (10/dose group) were exposed with DBA in drinking water at 0, 5, or 50 mg/kg/day from gestation day 15 though nursing. Upon weaning at 3 weeks, one group of pups (pre-pubertal group: 7-10 pups of each gender/treatment group) were euthanized and weights of liver, paired kidneys, testes, and ovaries were measured. In the 50 mg dose group, weights of testes and liver in males and weights of liver and kidneys in females were significantly higher (p < 0.05). The remaining pups (15-17 of each gender/dose group) continued to be dosed similarly through adulthood. At 7 weeks of age (neo-pubertal group), animals were euthanized and tissues weighed and processed for evaluation of reproductive organs and gametogenic potential. Except for decreased (p < 0.05) testes and kidney weights in 50 mg dose group males, there were no differences in organ weights. No significant differences were noted between control and dosed animals in daily sperm production, testicular sperm counts, epididymal sperm reserves, morphology of seminiferous epithelium, or ovarian follicle counts.

Expression of growth and differentiation factor-9 in the ovaries of fetal sheep homozygous or heterozygous for the inverdale prolificacy gene (FecX(I)).

Abnormal follicular and oocyte growth in ovaries of sheep homozygous (II) for the Inverdale gene, FecX(I), suggest that this gene may influence a fundamental event in initiation of folliculogenesis, with two copies of the gene inhibiting growth at the primordial/primary stage. In addition, striking similarities in ovarian morphology between mice deficient in growth and differentiation factor-9 (GDF-9) and II sheep suggest a relationship between the FecX(I) gene and GDF-9 function in the ovary. Therefore, it was hypothesized that GDF-9 mRNA expression would be inhibited in ovaries of II fetal sheep. To test this hypothesis, in situ hybridization was used to characterize GDF-9 mRNA expression in ovaries of homozygous (II), heterozygous (I+), and control (++) fetal sheep at Day 135 of gestation. GDF-9 mRNA expression was localized exclusively to oocytes from the type 1 follicle stage onward in all genotypes and is the first demonstration of GDF-9 mRNA expression in ovaries of fetal sheep. In addition, GDF-9 mRNA expression was detected in oocytes of abnormal type 2 follicles in the ovaries of II sheep. Thus, it does not appear that inhibition of GDF-9 gene expression is the mechanism of action whereby the FecX(I) gene exerts its influence. However, the possibility of translation at specific stages of follicular development cannot presently be ruled out. In addition, the FecX(I) gene may be involved, either directly or indirectly, in regulating expression of receptors for GDF-9. At present, however, neither the FecX(I) gene product nor the GDF-9 receptor has been isolated or characterized.

Effect of dose of prostaglandin F(2alpha) on steroidogenic components and oligonucleosomes in ovine luteal tissue.

To determine whether prostaglandin (PG) F(2alpha) had a dose-dependent effect upon secretion of progesterone, oligonucleosome formation, or loss of luteal weight, ewes on Day 9 or 10 of the estrous cycle were administered 0, 3, 10, or 30 mg PGF(2alpha) per 60 kg BW (i.v.), and luteal tissue was collected 9 and 24 h after injection. All doses of PGF(2alpha) decreased (P < 0. 05) concentrations of progesterone in sera by 9 h; however, in ewes treated with 3 mg PGF(2alpha), concentrations of progesterone were similar to control values at 24 h and higher (P < 0.05) than those in the 10- or 30-mg groups. Concentrations of progesterone in sera over all dose levels were highly correlated to luteal concentrations of mRNA encoding steroidogenic acute regulatory protein (P < 0.001), cytochrome P450 side-chain cleavage (P < 0.02), and 3beta-hydroxysteroid dehydrogenase (P < 0.01). Corpora lutea collected at 24 h from ewes treated with the 10- and 30-mg doses of PGF(2alpha) weighed less (P < 0.05) than those from controls. Oligonucleosomes were not present in luteal tissues from control ewes. Surprisingly, all doses of PGF(2alpha)-induced oligonucleosomes in a majority of animals at 9 h and in a majority of ewes treated with 10 and 30 mg of PGF(2alpha) at 24 h. In conclusion, 3 mg of PGF(2alpha) per 60 kg BW transiently decreased serum concentrations of progesterone and induced oligonucleosome formation, but did not result in reduced luteal weight. The 10- and 30-mg doses of PGF(2alpha) decreased secretion of progesterone and induced oligonucleosome formation and luteolysis.

Carcinoma in situ and seminoma in equine testis.

The presence of atypical germ cells resembling carcinoma in situ of human testis is reported for the first time in an unilaterally cryptorchid stallion. These cells were found in association with developing intratubular seminoma indicating they represented carcinoma in situ.

Steady-state luteinizing hormone receptor messenger ribonucleic acid levels and endothelial cell composition in bovine normal- and short-lived corpora lutea.

The short-lived corpus luteum (CL) contributes to reproductive inefficiency during the postpartum period in beef cows. The cause for the early demise of the short-lived CL is not fully understood but is believed to involve a premature release of prostaglandin F2 alpha. The objectives of this study were to evaluate norgestomet-hCG-induced normal-lived CL and hCG-induced short-lived CL in postpartum cows with respect to serum progesterone (P4) and 13,14-dihydro-15-keto, prostaglandin F2 alpha (PGFM) concentrations and luteal LH receptor (LH-R) concentrations, LH-R mRNA levels, and vascularity. Although serum P4 profiles from the time of hCG administration (Day 0) until luteectomy (Day 6, 7, or 8) were similar between CL life span groups, PGFM concentrations were elevated (p < 0.05) on Day 8 in cows expected to have short-lived CL compared to normal-lived CL. The LH-R concentrations were similar between normal- and short-lived CL on all days measured. Irrespective of luteal life span and day of luteectomy, all CL possessed a 4.4-kb LH-R transcript. Actin-normalized LH-R mRNA levels were similar between normal- and short-lived CL on Days 6 and 7; however, Day 8 short-lived CL contained less (p < 0.05) LH-R mRNA than Day 8 normal-lived CL. Although the area of luteal tissue occupied by capillaries in normal- and short-lived CL was similar on Days 6 and 7, the area occupied by capillaries in short-lived CL was lower (p < 0.05) than that for normal-lived CL on Day 8. Collectively, these results indicate that there is a decrease in steady-state LH-R mRNA and a reduction in luteal vascularity in CL expected to be short-lived. These changes occur concomitantly with a rise in serum PGFM, but prior to a decline in serum P4.

Use of semen as biopsy material for assessment of health status of the stallion reproductive tract.

Conventional light microscopic evaluation does not fully utilize potential indicators in seminal ejaculates for diagnosis of disorders of the reproductive tract. The technique of evaluation of all cellular components of semen, as described in this article, utilizing both light and transmission electron microscopy is a valuable diagnostic tool. Compare with other common biopsy procedures, use of semen as biopsy material is noninvasive, more representative than excisional biopsy, less expensive, and helps in the longitudinal evaluation after a therapeutic regimen.

An increase in serum lipids increases luteal lipid content and alters the disappearance rate of progesterone in cows.

To determine whether an increase in serum lipids alters the area occupied by lipid droplets in steroidogenic luteal cells and(or) clearance rates of progesterone from serum, pregnant beef heifers received control (n = 6) or treatment (n = 5) diets. To increase serum lipids, the treatment diet contained calcium soaps of fatty acids. Control and treatment diets were formulated to be isocaloric and isonitrogenous. Feeding of diets was initiated approximately 100 d before parturition and continued through the third postpartum estrous cycle. On d 12 or 13 of the third postpartum cycle, corpora lutea were collected by ovariectomy and a center slice was processed for electron microscopy. Eight samples from each slice were sectioned, stained, and examined at a magnification of 2,500x. Five micrographs per sample were analyzed for area occupied by small (SLC) and large (LLC) luteal cells, percentage of the area of each steroidogenic cell type occupied by lipid, and total steroidogenic area (SLC + LLC) occupied by lipid. Jugular blood was collected before and after ovariectomy, and progesterone, cholesterol, high-density lipoprotein (HDL), and low-density lipoprotein (LDL) were quantified. Cows consuming treatment diets had approximately twice (P < .05) the concentration of cholesterol, HDL, and progesterone in serum that controls had. The percentage of the area of SLC, LLC, and total area occupied by lipid was greater (P < .05) in treated than in control cows. The average time required for serum concentrations of progesterone to decrease by 50% after ovariectomy was greater (P < .05) in treated than in control cows (170 +/- 16 vs 113 +/- 15 min).(ABSTRACT TRUNCATED AT 250 WORDS)

Gonadotrope- and thyrotrope-specific expression of the human and bovine glycoprotein hormone alpha-subunit genes is regulated by distinct cis-acting elements.

The proximal 5'-flanking region of the alpha-subunit gene from humans and cattle confers pituitary-specific expression to heterologous reporter genes in transgenic mice. To investigate whether these promoter regions also contain the necessary regulatory elements for cell-specific expression and hormonal regulation, we used three independent lines of transgenic mice. Two lines of transgenic mice contained chimeric genes consisting of either 1.6 kilobasepairs (kbp) of human or 3 15 basepairs of bovine alpha-subunit proximal 5'-flanking sequence linked to the bacterial gene encoding chloramphenicol acetyltransferase (CAT). A third line of transgenic mice contained the proximal 1.6 kbp of 5'-flanking sequence of the human alpha-subunit gene linked to the bacterial lacZ gene encoding beta-galactosidase (beta gal; H alpha beta gal transgenic mice). Hormonal replacement paradigms indicate that both human and bovine alpha CAT transgenes are regulated by GnRH, suggesting that their expression occurs in gonadotropes. Thus, the proximal 5'-flanking regions of both the human and bovine alpha-subunit genes must contain regulatory elements that confer both gonadotrope-specific expression and responsiveness to GnRH. In contrast to the human alpha-subunit promoter, the bovine alpha-subunit promoter lacks a functional cAMP response element, suggesting that transduction of both cell-specific and GnRH transcriptional signals occurs through cAMP response element-independent pathways. Thyrotropes also express the glycoprotein hormone alpha-subunit gene. Yet, hormone replacement paradigms with propylthiouracil and T3 were ineffective in altering CAT activity in the pituitary of human or bovine alpha CAT transgenic mice. Because a thyroid hormone response element has been localized to the proximal 5'-flanking region of the human alpha-subunit gene, these data suggest that the alpha CAT transgenes lack sufficient information to direct expression to thyrotropes. Direct evidence for this possibility was obtained through immunocytochemical studies performed on pituitaries from H alpha beta gal transgenic mice. beta-Galactosidase activity appeared in gonadotropes, but not thyrotropes. We conclude, therefore, that distinct and separable regulatory elements mediate the expression of the alpha-subunit gene in gonadotropes and thyrotropes.

Numbers of steroidogenic luteal cells in Booroola Merino ewes.

In Exp. 1 ovulation rates, plasma concentrations of progesterone, mean individual and total CL weights were determined on Days 4, 10 and 12 after oestrus of Booroola Merino ++ ewes and FF ewes. Mean ovulation rates ranged from 1.5 to 1.8 in ++ ewes and from 5.3 to 6.2 in FF ewes (P less than 0.01). There were no differences in plasma concentrations of progesterone or total luteal weight between the two groups on any of the days studied. Individual CL were smaller (P less than 0.01) in FF ewes than in ++ ewes. In Exp. 2 the numbers of luteal cells in CL collected from 5 ++ and 5 FF ewes on Day 10 of the oestrous cycle were morphometrically determined. The CL from FF ewes were smaller (P less than 0.01) and had fewer total steroidogenic cells (P less than 0.01), fibroblasts (P less than 0.01), and capillary endothelial cells and pericytes (P less than 0.05). However, the luteal cell volume density, number of cells/g tissue, average cell diameter or average cell volume was not different between the two groups of ewes for any cell type studied. It is concluded that the 5-6 CL in FF ewes function in an identical fashion to the 1-2 CL in ++ ewes.

Functional and morphological characteristics of the first corpus luteum formed after parturition in ewes.

The ability of sheep luteal cells from the first corpus luteum formed after parturition (Group F) to secrete progesterone in the presence or absence of LH was compared with that of luteal cells obtained from normal cyclic ewes (Group C). Luteal concentrations of receptors for LH and prostaglandins (PG) F-2 alpha (PGF-2 alpha) and the cellular composition of corpora lutea from Groups F and C were also compared. Luteal cells from Group F secreted less progesterone in either the presence or absence of LH (P less than 0.01). There was no difference in the number of receptors for LH or PGF-2 alpha per luteal cell between Groups F and C (P greater than 0.1), nor was there a difference in the number of large or small steroidogenic luteal cells (P greater than 0.1). It was concluded that, if short-lived corpora lutea are insensitive to gonadotrophins, this response is not mediated by decreased numbers of receptors for LH. In addition, if the first corpus luteum formed post partum in ewes is more sensitive to the luteolytic effects of PGF-2 alpha, this effect is not mediated by an increased number of receptors for PGF-2 alpha or an increased proportion of PGF-2 alpha-sensitive large luteal cells.

Concentration of receptors for estradiol and progesterone in canine endometrium during estrus and diestrus.

Receptors for estrogen and progesterone were measured in cytosols prepared from specimens of canine endometrium obtained at late proestrus, day 4 of estrus, day 2 of diestrus, and at 10 day intervals from days 10 through 80 of diestrus. Twenty nine adult bitches were used, with 2 to 4 dogs used at each time point. Concentrations of estradiol receptors measured in endometrial cytosols from late proestrus through day 10 of diestrus were similar (mean +/- SEM: 9.9 +/- 2.2, 10.5 +/- 1.2, 16.3 +/- 1.6, and 16.2 +/- 2.9 pmol/g of tissue at proestrus, day 4 of estrus, days 2 and 10 of diestrus, respectively). As serum concentrations of progesterone increased during early diestrus, the concentration of estradiol receptors decreased and were significantly (P less than 0.05) lower on days 30 (4.9 +/- 1.3 pmol/g of tissue) and 40 (3.7 +/- 0.6 pmol/g of tissue) of diestrus. After day 40 of diestrus, when serum concentrations of progesterone were approaching basal concentrations, the concentration of estradiol receptors increased and remained significantly (P less than 0.05) higher from days 60 to 80 of diestrus (day 60, 13.4 +/- 2.9; day 70, 15.7 +/- 1.7; day 80, 19.8 +/- 2.4 pmol/g of tissue). As observed for estrogen receptors, the concentration of endometrial receptors for progesterone also gradually increased from late proestrus (4.9 +/- 1.3 pmol/g of tissue) to day 2 of diestrus (6.4 +/- 0.3 pmol/g of tissue).(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of luteinizing hormone and human chorionic gonadotropin on cell populations in the ovine corpus luteum.

Two experiments were conducted to examine the effect of treatment with human chorionic gonadotropin (hCG) or ovine luteinizing hormone (LH) on the number and size distribution of steroidogenic luteal cells. In Experiment I, 27 ewes were assigned to one of three groups: 1) hCG (300 IU, i.v.) administered on Days 5 and 7.5 of the estrous cycle (Day 0 = Estrus); 2) LH (120 micrograms, i.v.) administered at 6-h intervals from Days 5 to 10 of the cycle; 3) saline (i.v.) administered as in the LH treatment group. Blood samples were drawn daily from the jugular vein for quantification of progesterone. On Day 10, corpora lutea were collected, decapsulated, weighed, and dissociated into single cell suspensions. Cells were fixed, stained for 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) activity, and the size distribution of 3 beta HSD-positive cells was determined. Treatment with hCG, but not LH, increased (p less than 0.05) concentrations of progesterone in serum and the weight of corpora lutea. Treatment with either hCG of LH increased the proportion of cells greater than 22 micron in diameter and decreased the proportion of cells less than or equal to 22 micron (p less than 0.01). The ratio of small to large luteal cells decreased after treatment with either hCG or LH (p less than 0.05). In Experiment II, 9 ewes were assigned to one of two groups: 1) LH (120 micrograms, i.v.) administered at 6-h intervals from Days 5 to 10 of the estrous cycle, and 2) saline (i.v.) administered as in the LH treatment group.(ABSTRACT TRUNCATED AT 250 WORDS)

Luteal function in the bitch: changes during diestrus in pituitary concentration of and the number of luteal receptors for luteinizing hormone and prolactin.

The concentration of unoccupied luteal receptors for luteinizing hormone (LH) and prolactin, and the concentration of these two hormones in the pituitary was determined in 11 groups of bitches (n = 3 or 4/group) representing stages from proestrus through Day 80 of diestrus. Despite dramatic changes in serum concentrations of progesterone, the concentration of luteal receptors for LH and prolactin was quite constant throughout the entire luteal phase. In association with the ovulatory surge of LH, pituitary concentration of LH decreased abruptly from proestrus to Day 2 of diestrus, and was then gradually replenished during the remainder of diestrus. The concentration of prolactin in the pituitary did not vary significantly from proestrus through late diestrus.

Functional changes in luteinizing hormone-secreting cells from pre- and postpartum ewes.

Luteinizing hormone (LH)-containing cells from ovine pituitaries obtained during gestation and at various times after parturition were examined to determine whether the ability to store and secrete LH in vitro was correlated with morphological changes. Pituitaries collected on days 50 and 140 of gestation and on days 2, 13, 22, and 35 after parturition were enzymatically dissociated and the resulting cells cultured in media containing estradiol (12 pg/ml), cortisol (12 ng/ml), or no steroid. After 4, 7, or 10 days of culture, cells were washed and basal LH, gonadotropin-releasing hormone (GnRH)-stimulated release of LH, and cellular content of LH were determined. The content of LH (ng/10(6) cells) was lowest on day 140 of gestation (2.7 +/- 0.3) and day 2 postpartum (2.2 +/- 0.6) and then increased (P less than 0.05) on days 13 (36.6 +/- 8.3), 22 (59.9 +/- 14.4), and 35 (54.6 +/- 19.3) postpartum. The percentage of pituitary cells containing immunoreactive LH nearly doubled (P less than 0.05) between days 2 (5.6 +/- 0.2%) and 35 (10.6 +/- 1.1%) postpartum. Moreover, LH-containing cells were smaller, and the percent total cellular volume occupied by secretory granules was less on day 2 than on days 22 and 35 after parturition. Secretion of LH after 4, 7, or 10 days of culture reflected the cellular content of LH and was not influenced by the presence of steroids in the media. These data indicate that decreased synthesis of LH during gestation is associated with hypoplasia of the LH-secreting cells. These cells are reactivated during the postpartum period and their capacity to synthesize LH gradually returns to normal.

Role of luteinizing hormone in regulating luteal function in ruminants.

The technique of hypothalamic-pituitary stalk-disconnection was used to reinvestigate the roles of luteinizing hormone (LH) and prolactin in the regulation of luteal function in ewes. Stalk-disconnection was performed on d 5 of the estrous cycle and ewes were administered either saline (control), LH at 40 micrograms at 4-h intervals, 2 mg of alpha-ergocryptine at 12-h intervals or both LH and ergocryptine. The treatment regimen for LH was designed to mimic luteal phase concentrations of this hormone. Blood samples were collected from all stalk-disconnected and 6 sham-disconnected ewes at 4-h intervals beginning at 0600 h on the day of surgery for determination of serum concentrations of prolactin, cortisol and progesterone. Corpora lutea were collected from control ewes on d 5 of the estrous cycle and from the stalk-disconnected and sham-disconnected ewes on d 12 of the cycle. The luteal tissue was weighed, a slice taken for morphometric analysis of cell numbers, sizes and types and luteal progesterone content was determined. The weight and progesterone content of corpora lutea collected from stalk-disconnected ewes were similar to those observed in control ewes on d 5 of the cycle but less (P less than .05) than those in control ewes on d 12 of the cycle. However, serum concentrations of progesterone were unaffected by stalk-disconnection. Luteinizing hormone replacement therapy increased both the weight and progesterone content of corpora lutea in stalk-disconnected ewes to values similar to those observed in control ewes on d 12. Treatment of stalk-disconnected ewes with alpha-ergocryptine reduced serum concentrations of prolactin by greater than 95% but was without effect on the parameters of luteal function measured. The number of small steroidogenic luteal cells in any of the stalk-disconnected ewes was not different from that observed in control ewes. However, treatment of stalk-disconnected ewes with LH was followed by an increase (P less than .05) in the diameter of small luteal cells. The number of large luteal cells was greater (P less than .05) in LH-treated, stalk-disconnected ewes than in intact control ewes on d 12 of the estrous cycle. The mean diameter of large luteal cells was not affected by treatment with LH.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of progesterone on the oviductal epithelium in estrogen-primed prepubertal beagles: light and electron microscopic observations.

A total of 45 prepubertal beagles 6 to 8 weeks of age were used to study the cytological changes that accompany regression of the oviductal epithelium. The oviductal epithelium in untreated pups consisted of undifferentiated low cuboidal cells that measured 10.3 +/- 2.0 microns in height. In response to estradiol (E2), low cuboidal cells underwent hypertrophy, hyperplasia, and cytodifferentiation and gave rise to columnar ciliated and secretory cells. After 12 days of E2 treatment the epithelium was fully differentiated and measured 29.4 +/- 2.6 microns in height with 56% of the cells possessing cilia. When E2 treatment was continued for an additional 12 days, the epithelium was maintained in a differentiated state. However, if E2 treatment was terminated or progesterone (P) given alone or in conjunction with E2, the oviductal epithelium regressed and after 6 days was composed of low cuboidal cells that ranged in height from 9 to 14 microns with approximately 25% of the cells possessing cilia. A variety of cytological changes characterized the process of regression. The most immediate signs that regression was underway was a reduction in the height of the epithelium and the presence of cells with shrunken, pleomorphic nuclei that lacked prominent nucleoli. Degenerative events included: pinching off and shedding of the apical cytoplasm of cells comprising the epithelium, extrusion of whole cells and/or nuclei, and resorption of cilia and basal bodies. During the first 6 days following E2 withdrawal or P treatment, macrophages and cellular debris were frequently present within the lumen of the oviduct. The process of regression did not proceed synchronously throughout the ampulla of the oviduct, nor did all cells appear to degenerate in the same manner. The cytological changes that accompanied oviductal regression following P treatment were identical to those observed following E2 withdrawal. Results from experiments conducted in the present study show that: E2 induces the oviductal epithelium to differentiate and is required to maintain the epithelium in a differentiated state, E2 withdrawal or P treatment causes the oviductal epithelium to regress, at least three distinct degenerative processes are involved in the transition of columnar ciliated and secretory cells into low cuboidal cells, and regression does not occur synchronously throughout the ampulla region of the oviduct.