Porcine ovarian cells express messenger ribonucleic acids for the acid-labile subunit and insulin-like growth factor binding protein-3 during follicular and luteal phases of the estrous cycle.

It has recently been shown that, in follicular fluid, as in the circulation, insulin-like growth factors (IGFs)-I and -II exist in a ternary complex with IGF binding protein-3 (IGFBP-3) and the acid-labile subunit (ALS). The current study was designed to determine whether ovarian follicular and luteal cells could synthesize IGFBP-3 and ALS. Ovaries were collected, during the follicular and early luteal phases, from mature pigs whose cycles were synchronized with PGF2alpha. We studied IGFBP-3 and ALS messenger RNA (mRNA) by in situ hybridization. These transcripts were colocalized with aromatase mRNA, a marker of healthy granulosa cells. IGFBP-3 mRNA was equally expressed in granulosa cells of all growing follicles. In contrast, granulosa cell ALS mRNA levels were higher (P < 0.05) in preantral and small antral follicles than in large antral follicles. In thecal cells, expression of mRNA for IGFBP-3, ALS and cyclin D1 (a marker of cell proliferation) was restricted to healthy (aromatase-expressing) follicles. In those follicles, thecal expression of IGFBP-3 mRNA was low or absent in preantral follicles but increased (P < 0.05) in antral follicles. Thecal cell ALS transcripts peaked in small antral follicles (P < 0.05) and then declined. In granulosa cells of atretic follicles, transcripts for aromatase were greatly reduced, whereas IGFBP-3 mRNA levels remained high. In contrast, ALS transcript levels were greatly reduced in both granulosa (P < 0.05) and thecal cells (P < 0.001) of atretic follicles. After ovulation, IGFBP-3 mRNA was moderately expressed in granulosa luteins but strongly detected in a few theca-derived cells and in vascular endothelial cells. This study demonstrates that follicular fluid IGFBP-3 and ALS, like the IGFs, originate (at least in part) from the ovary. The ability of follicular cells to synthesize, assemble, and store all components of the ternary complex may be critical in determining the bioavailability of follicular IGF-I and -II.

Messenger ribonucleic acids for MAC25 and connective tissue growth factor (CTGF) are inversely regulated during folliculogenesis and early luteogenesis.

Cell proliferation, terminal differentiation, and angiogenesis occur during cycles of follicular and luteal development. In other paradigms, mac25, a potent tumor inhibitor is strongly induced in senescent epithelial cells, whereas CTGF stimulates angiogenesis and wound healing. Using in situ hybridization and immunohistochemistry, we have examined the possibilities that mac25 is inhibited, whereas CTGF is induced during active periods of follicular development and luteogenesis. Ovaries were collected during the follicular and early luteal phases from prostaglandin F2alpha-treated mature pigs and from slaughterhouse sows. CTGF transcripts were induced during the late preantral stage in granulosa and theca cells concomitantly with the appearance of endothelial cells in the theca. CTGF mRNA expression increased in granulosa cells to a maximum (P < 0.01) in mid-antral follicles but was down regulated (P < 0.01) in preovulatory follicles. In contrast, granulosa cell mac25 mRNA expression was undetectable between the preantral and mid-antral stage but was strongly induced in terminally differentiated granulosa cells of preovulatory follicles. CTGF mRNA and peptide were also detected in the theca externa/interstitium and in vascular endothelial cells of ovarian blood vessels, whereas mac25 transcripts, which were also abundant in ovarian blood vessels increased in the theca interna with follicular development. Transcripts of cyclin D 1, a marker of cell proliferation, appeared during the early antral stage and were moderate in granulosa cells but abundant in capillary endothelial cells in the theca interna, underneath the basement membrane. Following ovulation, CTGF and cyclin D1 mRNAs were associated with the migration of endothelial cells into the CL. Subsequently, there was a marked up-regulation of CTGF mRNA expression in granulosa luteins concomitantly with an increase in endothelial cell proliferation within the CL. We hypothesize that CTGF may promote ovarian cell growth and blood vessel formation during follicular and luteal development whereas mac25, a tumor inhibitor, may promote terminal differentiation of granulosa cells in preovulatory follicles.

Expression of mouse ovarian insulin growth factor system components during follicular development and atresia.

Insulin growth factor I (IGF-I) appears necessary for the completion of follicular development in mice. However, little is known about changes in the IGF system components during follicular development and luteinization. This study determined the relation between gene expression of specific IGF system components and follicular growth, survival, or atresia in mice. Immature mice from three different strains (129, C57, and MF1), with or without gonadotropin treatment (2.5 IU PMSG/2.5 IU human CG (hCG)], were used. The strains were similar in all parameters measured. Apoptosis, as detected by in situ labeling of nicked DNA, preceded the appearance of morphological signs of atresia. In healthy follicles, IGF-I transcripts were low during the primary follicular stage but increased to a maximum in the late preantral and early antral stages (P < 0.001) irrespective of hormone treatment. Occasionally, IGF-I transcripts were also detected in apoptotic follicles but decreased (P < 0.05) as a function of atresia as assessed by morphological criteria. IGF binding protein-4 (IGFBP-4) messenger RNA (mRNA) expression in granulosa cells was restricted to apoptotic and atretic follicles (P < 0.001). IGFBP-5 transcript levels, on the other hand, were elevated in granulosa cells of healthy primary and secondary follicles but decreased in subsequent follicular stages and in atretic follicles (P < 0.001). Conversely, IGFBP-2 mRNA was constitutively expressed in granulosa cells. PMSG/hCG treatment induced the appearance of IGFBP-2 transcripts in the ovarian interstitium. Following PMSG/hCG-induced ovulation, IGFBP-2 and -4 and IGF type-I receptor mRNAs were strongly expressed in virtually all luteal cells, whereas IGFBP-3 and -5 transcripts were selectively localized to some cell types in the corpus luteum. Conversely, IGF-I mRNA was essentially undetectable in the corpus luteum. This study represents the most comprehensive and detailed analysis of the physiology and anatomy of the mouse ovarian IGF system, and shows that 1) IGFBP-5-is linked to the survival of the slow growing and immature preantral follicles; 2) IGF-I is associated with the growth and survival of the rapidly growing large preantral and antral follicles; 3) IGFBP-4 is an atretogenic candidate for mouse ovarian follicles; 4) ovulatory doses of PMSG/hCG up-regulate IGFBP-2 mRNA expression in the ovarian interstitium; and 5) transcripts of IGF type-I receptor and IGFBP-2 through -5, but not those of IGF-I are highly expressed in the mouse corpus luteum.

Activation of bovine and baboon primordial follicles in vitro.

Mammalian ovaries contain a large pool of non-growing, primordial follicles. The ability to initiate growth of this pool of resting follicles in vitro and to maintain follicular growth to a stage when the oocyte could be matured and fertilized would increase the reproductive potential of valuable domestic animals, endangered species and infertile women. This paper summarizes our progress to date in activating primordial follicles of cattle and baboons. Pieces of ovarian cortex, rich in primordial follicles, were obtained from fetal bovine and baboon ovaries during late gestation. Pieces were maintained in organ culture in serum-free medium containing ITS+ (insulin-transferrin-selenium-linoleic acid-BSA) for up to 20 days and at various times during culture some pieces were fixed for histological morphometry. As early as 2 days of culture, the number of primordial follicles had decreased by 88% or 55%, whereas the number of primary follicles had increased 2.5- or 5-fold, compared to tissue freshly isolated from bovine or baboon ovaries, respectively (P < 0.01). In baboon cortical pieces a small number of secondary follicles developed during a 20-day culture period. The development of primary and secondary follicles was accompanied by an increase in diameter of both the granulosa cell layer and the oocyte. The addition of FSH (1, 10, or 100 ng/ml) had no effect on the development of follicles in bovine cortical pieces after 7 or 14 days of culture, relative to control cultures without FSH. These results show that a high percentage of primordial follicles from cattle and baboons can be activated to grow in serum-free medium in the absence of gonadotropins. Conditions that will support further growth in vitro of follicles from these species remain to be elucidated. The culture system we have developed could be used to develop such conditions and to explore factors that regulate the movement of primordial follicles into the pool of growing follicles.

Initiation of growth of baboon primordial follicles in vitro.

Factors that cause some primordial follicles to enter the growth phase while the others remain quiescent are unknown. The hypothesis was tested that primate primordial follicles can survive and initiate growth in vitro in serum-free medium. Superficial pieces of ovarian cortex, containing mostly primordial follicles, were obtained from baboon fetuses during late gestation and cultured for 0, 2, 4, 7, 10 or 20 days in Waymouth MB 752/1 medium supplemented with insulin, transferrin, selenium, linoleic acid, and bovine serum albumin (ITS +). Histological examination of cortical pieces revealed that after 2 and 4 days in culture, the total number of primordial follicles had decreased by 55 and 76% (P < 0.01) respectively, relative to day 0 of culture. This was associated with a sustained, 5- to 8-fold increase in total primary follicles (P < 0.01) beginning on day 2 of culture. There was also a gradual increase in the total number of early secondary and secondary follicles. The average diameter of follicles and oocytes increased gradually throughout culture for all follicular categories (P < 0.01), except secondary follicles and oocytes. Immunohistochemical localization of proliferating cell nuclear antigen (PCNA), a marker for cell proliferation and growth, showed that PCNA was generally absent in primordial follicles on day 0, but was observed after 2 or 4 days in culture in both granulosa cells and oocytes of most growing follicles. Comparison of cortical pieces cultured for 10 or 20 days with ITS + versus 10% fetal bovine serum (FBS) showed a more pronounced decrease in the numbers of primordial follicles and more primary, early secondary and secondary follicles in ITS + compared to FBS-treated cortical pieces (P < 0.01 at 20 days). These results show that primordial follicles from non-human primates can survive and develop to the secondary stage in vitro in serum-free conditions.

Changes in androgen secretion and luteinizing hormone pulse amplitude are associated with the recruitment and growth of ovarian follicles during the luteal phase of the bovine estrous cycle.

In cattle the development of large antral follicles occurs in two or three successive waves during the estrous cycle, with one follicle per wave selected for dominance. To test the hypothesis that negative feedback effects of steroids secreted by the dominant follicle are critical to the regulation of follicular waves, we examined temporal relationships among ovarian follicular growth, steroid secretion, and gonadotropin secretion. Follicular growth was monitored by ultrasonography. In the first experiment, blood was collected from 5 Holstein heifers every 8 h between Days 0 and 14 of the estrous cycle from both a jugular vein and the vena cava (to collect ovarian blood). Jugular samples were also collected every 12 min for 8 h during three periods (Days 3 or 4, 7 or 8, and 11, 12, or 13; n = 6) to characterize the pulsatile pattern of LH secretion. Both estradiol and testosterone concentrations in the vena cava increased as pre-wave elevations in FSH concentrations decreased (p < 0.05) between Days 1 and 4 (first follicular wave) and between Days 9 and 12 (second follicular wave). LH pulse amplitude was greater during the second period of frequent blood collection (Day 7 or 8, end of the growth phase of the first dominant follicle) compared to the other two periods (p < 0.05), suggesting that increased LH pulse amplitude may be important for the later stages of dominant follicle growth. In the second experiment, to determine whether ovarian steroids are secreted primarily by dominant follicles, blood samples were collected from the utero-ovarian veins draining ovaries with (n = 4) and without (n = 4) a dominant follicle during the first follicular wave. Testosterone, androstenedione, and estradiol concentrations in the utero-ovarian veins fluctuated in relation to the pattern of follicular growth (p < 0.05), and secretion was much greater from ovaries with a dominant follicle. In blood collected both from the vena cava and from the utero-ovarian veins, estradiol secretion reached a peak and started to decline before androgen concentrations peaked (p < 0.05), suggesting that the initial decrease in estradiol secretion from the dominant follicle is not due to a lack of androgen precursors. The results suggest that 1) a transient increase in LH pulse amplitude during the early-midluteal phase may be important for supporting the final stages of dominant follicle growth; 2) ovarian androgens, as well as estradiol, may play a critical role in the control of FSH secretion during waves of follicular development; and 3) the dominant follicle is responsible for fluctuations in circulating estradiol and androgens during follicular waves.

Initiation in vitro of growth of bovine primordial follicles.

Factors that control the onset of primordial follicle growth are unknown. We have tested the hypothesis that primordial follicles from fetal calves can survive and initiate growth in vitro in serum-free conditions. Superficial pieces of ovarian cortex, containing mostly primordial follicles, were isolated from bovine fetuses 6-8 mo old and cultured for 0, 2, 4, or 7 days in Waymouth MB 752/1 medium supplemented with insulin, transferrin, selenium, linoleic acid, and BSA (ITS+). Histological examination of cortical pieces after 2, 4, and 7 days in culture showed that the number of healthy primordial follicles had decreased by 88%, 90%, and 94%, respectively (p < 0.01), whereas the number of healthy primary follicles had increased to 260%, 209%, and 197%, respectively, of the number present on Day 0 (p < 0.05). The percentage of follicles that showed signs of atresia did not change with time in culture and was about 28% and 50% for primordial and primary follicles, respectively. After 7 days in culture, the mean diameter of the few remaining healthy primordial follicles was 1.2 times the average diameter of primordial follicles present on Day 0 (p < 0.01). In contrast, after 2, 4, and 7 days in culture, primary follicles were 1.2, 1.3, and 1.4 times larger in diameter, respectively, relative to Day 0 (p < 0.01). There was little change in the diameter of oocytes in primordial follicles during culture, whereas in primary follicles an increase in oocyte diameter became apparent after 4 and 7 days (1.1 and 1.2 times, respectively, p < 0.01). That follicle growth was initiated in vitro was further confirmed by immunolocalization of proliferating cell nuclear antigen (PCNA), a marker for cell growth and proliferation, in cultured and freshly isolated pieces of ovarian cortex. In freshly isolated tissue, PCNA staining was absent from pre-granulosa cells and oocytes of the quiescent primordial follicles but was intense in granulosa cells and oocytes of the few growing primary follicles. After 2, 4, and 7 days in culture, PCNA was expressed intensely in the oocyte and many granulosa cells of newly activated primary follicles. These results demonstrate that bovine primordial follicles can enter the growth phase in vitro and that PCNA expression by granulosa cells and oocytes is closely associated with the onset of primordial follicle growth. The fact that a high percentage of primordial follicles initiated growth in vitro suggests that the ovarian stroma exerts inhibitory control over the initiation of primordial follicle growth in vivo. The culture system we describe may provide the means to test this hypothesis and others.

FSH and growth factors affect the growth and endocrine function in vitro of granulosa cells of bovine preantral follicles.

The hypothesis was tested that bovine preantral follicles can be stimulated to grow in vitro by FSH and by the mitogens, epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF), but not by transforming growth factor-beta (TGFbeta), which generally inhibits EGF and bFGF action. Preantral follicles, 60 to 179 mum in diameter, were isolated from fetal ovaries by treatment with collagenase and DNase and cultured for 6 d in serum-free medium, with or without FSH and growth factors. Basic FGF (50 ng/ml), and to a lesser extent FSH (100 ng/ml) and EGF (50 ng/ml), stimulated thymidine incorporation by granulosa cells in bovine preantral follicles compared to control cultures (8-, 4- and 2.5-fold the labeling index of the controls; P < 0.05). Alone TGFbeta (10 ng/ml) had no effect on (3)H-thymidine incorporation, but it completely inhibited the bFGF- but not the FSH-stimulated increase in the labeling index and mean follicular diameter of preantral follicles (P < 0.05). By the end of the culture period oocytes in most treatments had degenerated, and the few surviving oocytes were in preantral follicles cultured with FSH or bFGF. Progesterone accumulation was greater (P < 0.05) in the presence of FSH (100 ng/ml) or EGF (50 ng/ml) than with bFGF, TGFbeta or control medium. Basic FGF strongly inhibited the effect of FSH on progesterone secretion (P < 0.05). Only FSH stimulated the conversion of exogenous testosterone to estradiol and both bFGF and TGFbeta markedly inhibited FSH-stimulated estradiol accumulation. These results indicate that proliferation of granulosa cells of bovine preantral follicles can be stimulated by bFGF, FSH and EGF, whereas TGFbeta inhibits growth, and that they are steroidogenically active in culture. Basic FGF and TGFbeta antagonize FSH-stimulated steroid production by granulosa cells of cultured bovine preantral follicles.

Ontogeny and cellular localization of 125I-labeled basic fibroblast growth factor and 125I-labeled epidermal growth factor binding sites in ovaries from bovine fetuses and neonatal calves.

We have recently reported that although specific 125I-FSH receptors are present in granulosa cells from primary and secondary follicles, gonadotropin responsiveness is very low in ovaries from bovine fetuses, which consist mainly of preantral follicles with few early antral follicles. It is well established that a number of polypeptide growth factors show pronounced mitogenic effects on follicular cells. Therefore, we have compared autoradiographically the ontogeny and cellular localization of basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) binding activities to assess their possible involvement in the regulation of early follicular growth in fetuses and neonatal calves. Follicular growth was initiated around Day 180 of gestation in fetuses. 125I-bFGF binding values were high in granulosa cells from preantral follicles (mean +/- SEM, 7.8 +/- 1.1-9.8 +/- 0.7 grains/cell, 0.05-0.15-mm diam.) but decreased in early antral follicles (0.16-3.0 mm) to a constant level (5.7 +/- 1.2 grains/cell). Specific 125I-EGF binding values were low in preantral follicles but showed a 2.5- and 5.0-fold increase in both granulosa cells and the theca interna from antral I (0.16-0.5 mm) and antral II follicles (0.6-3.0 mm), respectively. In atretic follicles, 125I-bFGF specific binding values were high (10.4 +/- 0.8 grains/cell), whereas 125I-EGF binding levels were significantly reduced or absent. None of the radioligands tested bound significantly to primordial follicles. There was no age-related difference in any ligand binding to follicles of comparable size. These results provide novel evidence that bFGF, a potent mitogen, is involved in the regulation of granulosa cell function as early as the preantral stage in cattle.(ABSTRACT TRUNCATED AT 250 WORDS)

Ontogeny and cellular localization of 125I-labeled insulin-like growth factor-I, 125I-labeled follicle-stimulating hormone, and 125I-labeled human chorionic gonadotropin binding sites in ovaries from bovine fetuses and neonatal calves.

In a previous study we reported that ovaries from bovine fetuses, which consist mainly of preantral follicles with few antral follicles, are weakly responsive to gonadotropins (FSH and LH). Insulin-like growth factor-I (IGF-I) is known to enhance gonadotropin responsiveness in vitro, but there is a lack of consistent data on the involvement of IGF-I, FSH, and LH during early stages of folliculogenesis in cattle. In the study reported here, we assessed autoradiographically the ontogeny of 125I-gonadotropin and 125I-IGF-I binding activities during preantral and early antral stages in cattle. Follicular growth was initiated around Day 180 of gestation in fetuses. The density of 125I-FSH binding was high in granulosa cells from primary (mean +/- SEM 10.5 +/- 0.7 grains/cell, 0.05-mm diam.) and secondary follicles (10.8 +/- 0.8 to 13.6 +/- 1.2 grains/cell, 0.06-0.15 mm) but increased significantly (p < 0.05) in early antral follicles (18.2 +/- 1.1 grains/cell, 0.16-3.0 mm). Specific 125I-IGF-I binding levels were low in granulosa cells from preantral follicles, averaging 2.5 +/- 0.6-3.1 +/- 0.9 grains/cell. However, after antrum formation, the density of 125I-IGF-I binding increased significantly (p < 0.05) with follicular diameter in granulosa cells and was 5.7 +/- 0.7 and 9.1 +/- 0.6 grains/cell for antral I (0.16-0.5 mm) and antral II (0.6-3.0 mm) follicles, respectively. 125I-FSH and 125I-IGF-I binding densities were low in theca cells from preantral and early antral follicles as well as in the interstitial tissue and granulosa cells from atretic follicles.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential response to gonadotropins and prostaglandin E2 in ovarian tissue during prenatal and postnatal development in cattle.

In cattle, growing follicles are present in fetal ovaries during the last part of gestation. This study examines the extent of changes in basal and hormone-stimulated adenylyl cyclase (AC) activity in ovaries of the bovine fetus when the first follicles begin to grow. The first growing follicles appeared in fetal ovaries around Day 180 and consisted mainly of primary and secondary follicles; few antral follicles were present before Day 220 of gestation. Basal AC activity in ovarian membranes increased simultaneously with the beginning of follicle growth in the fetus (5.8 +/- 0.9 vs. 9.3 +/- 1.3 pmol cAMP/mg protein/min at 130-180 and 180-210 days of gestation, respectively p less than 0.05). During the same time period, there was a significant increase in both the absolute (16.1 +/- 1.2 to 39.9 +/- 1.4 pmol cAMP/mg protein/min) and the relative (2.8 +/- 0.1 to 4.3 +/- 0.3 times the basal level, p less than 0.05) effects of guanosine triphosphate (GTP). After birth, basal and GTP-stimulated AC activities (pmol cAMP/mg protein/min) increased markedly in ovarian membranes of 1-wk-old calves and then decreased with age; the lowest levels were measured in mature cyclic cows. However, the relative effect of GTP (times the basal level) did not show this age-related variation. Prostaglandin E2 (PGE2) stimulation of AC in ovarian membranes from fetuses was high even on Day 120 (2.1 +/- 0.3 times the control level).(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of intrahypothalamic injections of GABA, muscimol, pentobarbital, and L-glutamic acid on feed intake of satiated sheep.

Five wethers were surgically prepared with cranial implants to study the role of gabaminergic neural pathways on the hypothalamic control of feeding behaviour in ruminants. In the first experiment, the animals were injected (1 microL) with a physiological Tyrode (0.95%) solution, muscimol (0.5 and 1.0 nmol), GABA (0.5 and 1.0 nmol), and L-glutamic acid (0.5 and 1.0 nmol). Feed intake following injections of muscimol (1.0 nmol) and L-glutamic acid (0.5 and 1.0 nmol) was twice as large as that following the Tyrode solution, at 60-min postinjections. These results, however, were not statistically significant (p = 0.12-0.15). In the second experiment, the animals were injected (1 microL) with saline, muscimol (0.8 nmol), L-glutamic acid (0.8 nmol), and pentobarbital (0.26 mumol). Fifteen minutes after the injections, pentobarbital had induced a significant feeding response when compared with control values (p less than 0.01), whereas the effect of L-glutamic acid was not significant. However, 30 min after the injections, feed intake of sheep having received L-glutamic acid was higher than that obtained with the control injections (p less than 0.01). The response to pentobarbital was stronger than that to either muscimol or L-glutamic acid. Histological analyses of brain tissue indicated that injections were performed in the ventromedial hypothalamus of four sheep and in the dorsomedial hypothalamus of the other. The data indicate that L-glutamic acid stimulates feed intake by acting either as a precursor of GABA or by a direct stimulation of glutaminergic neural pathways involved in the control of feed intake.