Physiology of GDF9 and BMP15 signalling molecules.

Two related oocyte-derived members of the transforming growth factor-beta (TGF-beta) superfamily, namely growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15, also known as GDF9B), have recently been shown to be essential for ovarian follicular growth. In addition, both proteins have been shown to regulate ovulation rate in sheep, and although it is evident that these growth factors interact both with one another and with other intra- and extra-ovarian factors, the precise mechanisms by which they influence follicular growth and ovulation rate have not been thoroughly elucidated.

Chronic exposure to dibromoacetic acid, a water disinfection byproduct, diminishes primordial follicle populations in the rabbit.

To determine if dibromoacetic acid (DBA) affects ovarian folliculogenesis, four groups of female Dutch-belted rabbits were exposed daily to 0, 1, 5, or 50 mg DBA/kg body weight in drinking water beginning in utero from gestation day 15 throughout life. Functionality of the endocrine axis was assessed by measuring serum concentrations of gonadotropins following an im injection of 10 microg GnRH at 12 (prepubertal; n = 6/dose group) and 24 (postpubertal; n = 10/dose group) weeks of age. A day after GnRH challenge, number of ovulation sites and ovarian weights were determined at necropsy. Left ovaries were processed for histopathology, serially sectioned at 6 microm, and every twelfth section stained with hematoxylin and eosin was evaluated. All healthy follicles were categorized as primordial, primary, small preantral, large preantral, or small antral follicles. The area of each section evaluated was measured and the number of follicles in each category expressed per mm2 unit area. In prepubertal animals, DBA caused a reduction in number of primordial follicles (p < 0.05) and total healthy follicles (p < 0.05) at 50 mg/kg dose level. In adult animals, there were fewer primordial follicles in both the 5 (p < 0.01) and 50 (p = 0.1) mg/kg dose groups. No profound changes in gonadotropin profiles were observed. Although chronic exposure to DBA did not appear to have an effect on late follicular development or ovulation, DBA did reduce the population of primordial follicles. The long-term health consequences of diminished primordial follicles are unknown, but it is very likely that reproductive senescence would occur earlier.

Oocyte-derived growth factors and ovulation rate in sheep.

The physiological mechanisms controlling ovulation rate in mammals involve a complex exchange of endocrine signals between the pituitary gland and the ovary, and a localized exchange of intraovarian hormones between the oocyte and its adjacent somatic cells. The discoveries in sheep of mutations in bone morphogenetic protein 15 (BMP15) and bone morphogenetic protein receptor type IB (BMPR-IB) together with recent findings on the physiological effects of growth differentiation factor 9 (GDF9) and BMP15 on follicular development and ovulation rate highlight some important differences in the way in which the oocyte may function in mammals with different ovulation rate phenotypes. In sheep, BMP15 and GDF9 have each been shown to be essential for the early and later stages of follicular development. In addition, ovulation rate is sensitive to changes in the dose of either of these two oocyte-derived growth factors. These findings are in contrast to those reported for mice in which GDF9, but not BMP15, is essential for follicular development. The evidence to date is consistent with the hypothesis that the oocyte plays a central role in regulating key events in the process of follicular development and hence, is important in determining ovulation rate. Moreover, it appears that the mechanisms that the oocyte uses to control these processes differ between species with low and high ovulation rate phenotypes.

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.

Molecular cloning of the ovine Growth/Differentiation factor-9 gene and expression of growth/differentiation factor-9 in ovine and bovine ovaries.

Recently a novel member of the transforming growth factor beta (TGFbeta) superfamily termed growth/differentiation factor-9 (GDF-9) was shown to be expressed in ovaries of mice and humans, and to be essential for normal follicular development beyond the primary (type 2) follicle stage in mice. In the present study, the gene for ovine GDF-9 was isolated and characterized, and expression of GDF-9 mRNA in ovaries of domestic ruminants was examined. The predicted amino acid sequence of ovine GDF-9 is 77% and 66% homologous to human and mouse GDF-9, respectively. Specific hybridization using homologous 35S-antisense probes was restricted to oocytes. In contrast to similar studies in mice in which GDF-9 was first detected beginning at the primary (type 2) follicle stage, in ovine and bovine ovaries GDF-9 mRNA was expressed beginning at the primordial (type 1) follicle stage. The observed timing and pattern of GDF-9 expression in oocytes of domestic ruminants is consistent with a role for GDF-9 in the initiation and maintenance of folliculogenesis in these species, and supports the general concept that early stages of follicular growth and development are regulated by intraovarian factors.

Localization and quantification of binding sites for follicle-stimulating hormone, luteinizing hormone, growth hormone, and insulin-like growth factor I in sheep ovarian follicles.

In sheep, growth and development of ovarian follicles beyond 2 mm in diameter is acutely dependent on gonadotropin support. As a consequence, following hypophysectomy (HPX) or hypothalamic-pituitary stalk disconnection (HPD), growth of follicles beyond 2 mm is arrested and all follicles > 2 mm undergo atresia. Although administration of exogenous gonadotropins stimulates follicular growth and ovulation in HPD ewes, follicles in HPX ewes remain unresponsive unless growth hormone (GH) is also given. To determine whether the difference in follicular sensitivity to gonadotropins after HPD (gonadotropin sensitive) or HPX (gonadotropin insensitive) is related to the distribution and quantity of binding sites for FSH, LH, and/or insulin-like growth factor I (IGF-I), binding sites for these hormones were localized and quantified using topical autoradiography in healthy follicles from control (pituitary-intact), HPD, and HPX ewes. In addition, in situ hybridization was performed to localize mRNA for GH and FSH receptors. Irrespective of treatment, binding of FSH and mRNA for FSH receptor were greatest (p < 0.05) in the membrana granulosa; LH binding was greatest (p < 0.05) in the theca interna; and IGF-I binding was greatest (p < 0.05) in the theca externa. Although the relative number of binding sites for LH did not differ among treatments, those for FSH and IGF-I were lower (p < 0.05) in HPD and HPX ewes compared to controls. Attempts to quantify binding sites for GH were unsuccessful due to high nonspecific binding. However, mRNA for GH receptor was most abundant (p < 0.05) in the membrana granulosa and oocytes of small antral and preantral follicles. Compared to levels in controls and HPD ewes, the level of GH receptor mRNA was lower (p < 0.05) in follicles obtained from HPX ewes. On the basis of these data, failure of small antral follicles in HPX ewes to respond to exogenous gonadotropins is not due to a reduction in receptors for FSH, LH, or IGF-I. The observed reduction of mRNA for GH receptor in the membrana granulosa of follicles from HPX ewes provides evidence that GH may play an important role in early stages of folliculogenesis and that it is involved in the maintenance of sensitivity to gonadotropins.

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)

Recombinant bovine somatotropin does not improve superovulatory response in sheep.

Although treatment of heifers and ewes with recombinant bovine somatotropin (rbST) does not increase ovulation rate, data for heifers indicate that the number of small antral follicles is approximately doubled. Accordingly, the objectives of this study were to determine whether 1) treatment of ewes with rbST would increase the number of small antral follicles, thereby increasing the number of follicles that could potentially respond to superovulation treatment, and 2) superovulatory responses could be improved in ewes with "synchronized" populations of follicles. Twenty-four ewes were divided into four groups: control, control+rbST, hypothalamic-pituitary stalk disconnected (HPD), and HPD+rbST. Beginning on d 5 of the estrous cycle, ewes were injected once daily for 13 d with either rbST (3 mg) or saline. The superovulatory regimen consisted of a single dose of PMSG followed by twice-daily injections of FSH for four consecutive days. After ovariectomy, ovulation sites and follicles were counted. Twice-daily blood samples were assayed for somatotropin (ST) and IGF-I. The concentrations of ST in rbST-treated ewes were greater (P < .05) than those in controls. Treatment with rbST increased (P < .05) the mean serum concentration of IGF-I in control but not in HPD ewes. There was no increase in ovulation rate or number of small antral follicles in response to rbST. Synchronizing follicle populations also failed to increase ovulation rate or reduce variability of response. We conclude that supplementation with rbST and synchronization of follicles does not increase the superovulatory response in sheep.

Proliferation of jejunal mucosal cells in rats flown in space.

To examine the effects of spaceflight on the proliferation and turnover of jejunal mucosal cells, we compared the percentages of mitotic cells present in the crypts of Lieberkühn in the proximal, middle, and distal jejunum in each of five rats flown on the COSMOS 2044 mission and in rats included in the vivarium, synchronous, and caudal-elevated groups. On the basis of the data obtained, there was no difference in mitotic indexes between animals in the flight and vivarium (ground control) groups. Thus it appears that the ability of jejunal mucosal cells to proliferate is not affected by microgravity conditions associated with spaceflight. Although the length of villi and depth of crypts were reduced in flight animals compared with those in the vivarium group, the observed reduction is probably attributable to changes in the connective tissue core of villi and is not likely due to an impairment of the proliferation and migration of jejunal mucosal cells.

Effects of spaceflight on the proliferation of jejunal mucosal cells.

The mitotic indices, villus heights, and crypt depths were determined in each of three jejunal regions (proximal, middle, and distal) for five animals each in the flight, vivarium, and synchronous groups. Because of the rapid turnover of intestinal mucosal cells and the delay in recovering the flight animals, it is not known whether the proliferation of jejunal mucosal cells is affected by microgravity conditions associated with spaceflight. However, since there were no consistent differences between animals in the flight group and those in the synchronous and vivarium control groups, it appears that any effects of microgravity on the turnover of jejunal mucosal cells are short-lived. Thus, this study represents an initial step in determining the effects of microgravity on the proliferation and turnover of intestinal mucosal cells.

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)

Morphometric analysis of cell types in the ovine corpus luteum throughout the estrous cycle.

The cellular composition of ovine corpora lutea obtained during the early (Day 4), mid (Days 8 and 12), and late (Day 16) stages of the estrous cycle was determined by morphometric analysis. Individual corpora lutea were collected via midventral laparotomy from a total of 19 ewes. A center slice from each corpus luteum was processed for electron microscopy and subsequent morphometric analysis of the numbers and sizes of steroidogenic and nonsteroidogenic cells. Luteal weight progressively increased throughout the estrous cycle (p less than 0.05). Corpora lutea collected on Day 16 were assigned to one of two subgroups on the basis of gross appearance and weight: nonregressed (NR, 542 +/- 25 mg) or regressed (R, 260 +/- 2 mg). There were no significant changes in the proportion of the corpus luteum occupied by small luteal cells (19 +/- 2%) or large luteal cells (36 +/- 1%) throughout the estrous cycle. The total number of steroidogenic cells per corpus luteum increased from 21.8 +/- 3.7 (X 10(6)) on Day 4 to 61.7 +/- 5.4 (X 10(6)) on Day 8 (p less than 0.05) and remained elevated thereafter. The number of small luteal cells was 10.0 +/- 2.7 (X 10(6)), 39.7 +/- 1.4 (X 10(6)), 46.1 +/- 5.8 (X 10(6)), 49.0 +/- 13.7 (X 10(6)), and 29.9 +/- 8.6 (X 10(6)) on Days 4, 8, 12, 16 (NR), and 16 (R), respectively (p less than 0.05, Day 4 vs. Days 8, 12, 16 NR). In contrast, the number of large luteal cells was 11.8 +/- 1.5 (X 10(6)) on Day 4 and did not vary significantly during the remainder of the estrous cycle. The numbers of nonsteroidogenic cell types increased (p less than 0.05) from Day 4 to Day 16 (NR) but were decreased in regressed corpora lutea (Day 16 R). Regression was characterized by a 50% decrease (p less than 0.05) in the total number of cells per corpus luteum from 243 +/- 57 ( X 10(6)) on Day 16 (NR) to 125 +/- 14 ( X 10(6)) on Day 16 (R) (p less than 0.05). Small luteal cells remained constant in volume throughout the entire estrous cycle (2520 +/- 270 microns 3), whereas large luteal cells increased in size from 5300 +/- 800 microns 3 on Day 4 to 16,900 +/- 3300 microns 3 on Day 16 (NR) (p less than 0.05). In summary, small luteal cells increased in number but not size throughout the estrous cycle, whereas large luteal cells increased in size but not number.

Immunocytochemical localization of neurophysin and oxytocin in ovine corpora lutea.

The cellular distribution of neurophysin and oxytocin within ovine corpora lutea obtained on Days 4, 10 and 16 of the estrous cycle was examined immunocytochemically. Serial sections (8-10 micron-thick) prepared from corpora lutea that had been fixed in Bouin's solution and embedded in paraffin were immunostained for neurophysin or oxytocin using the peroxidase-antiperoxidase (PAP) procedure. Irrespective of the day of the cycle examined, immunoreactivity was restricted to large luteal cells. However, on Days 4 and 10 of the cycle, the intensity of staining in large luteal cells was highly variable; and, within the same section some cells were heavily stained, others were only lightly stained, and still others were not stained at all. In contrast, on Day 16 of the cycle, the intensity of staining was uniform and essentially all of the large luteal cells were immunoreactive. Based on the results obtained, it is evident that immunoreactive neurophysin and oxytocin can be detected as early as Day 4 of the cycle, persists through Day 15, and is restricted to large luteal cells.