Cells Of Large Follicles Research Articles

Hormone-induced proliferation and differentiation of granulosa cells: a coordinated balance of the cell cycle regulators cyclin D2 and p27Kip1.

The proliferation and terminal differentiation of granulosa cells are critical for normal follicular growth, ovulation, and luteinization. Therefore, the in situ localization and hormonal regulation of cell cycle activators (cyclin D1, D2, and D3) and cell cycle inhibitors (p27Kip1 and p21Cip1) were analyzed in ovaries of mice and rats at defined stages of follicular growth and differentiation. Cyclin D2 mRNA was specifically localized to granulosa cells of growing follicles, while cyclin D1 and cyclin D3 were restricted to theca cells. In hypophysectomized (H) rats, cyclin D2 mRNA and protein were increased in granulosa cells by treatment with estradiol or FSH and were increased maximally by treatment with both hormones. In serum-free cultures of rat granulosa cells, cyclin D2 mRNA was rapidly elevated in response to FSH, forskolin, and estradiol, indicating that estradiol as well as cAMP can act directly and independently to increase cyclin D2 expression. The levels of p27Kip1 protein were not increased in response to estradiol or FSH. In contrast, when ovulatory doses of human CG (LH) were administered to hormonally primed H rats to stimulate luteinization, cyclin D2 mRNA and protein were rapidly decreased and undetectable within 4 h, specifically in granulosa cells of large follicles. Also in response to LH, the expression of the cell cycle inhibitor p27Kip1 was induced between 12 and 24 h (p21Cip1 was induced within 4 h) and remained elevated specifically in luteal tissue. A critical role for cyclin D2 in the hormone-dependent phase of follicular growth is illustrated by the ovarian follicles of cyclin D2-/- mice, which do not undergo rapid growth in response to hormones, but do express markers of FSH/LH action, cell cycle exit, and terminal differentiation. Collectively, these data indicate that FSH and estradiol regulate granulosa cell proliferation during the development of preovulatory follicles by increasing levels of cyclin D2 relative to p27Kip1 and that LH terminates follicular growth by down-regulating cyclin D2 concurrent with up-regulation of p27Kip1 and p21Cip1.

Differential responses of granulosa cells from small and large follicles to follicle stimulating hormone (FSH) during the menstrual cycle and acyclicity: effects of tumour necrosis factor-alpha.

This study determined effects of follicle stimulating hormone (FSH) alone and in combination with tumour necrosis factor (TNF), on granulosa cells from small (5-10 mm diameter) and large (>10-25 mm) follicles during follicular and luteal phases of the cycle and during periods of acyclicity. Granulosa cells were collected from ovaries of premenopausal women undergoing oophorectomy. The cells were cultured with human FSH (2 ng/ml) and testosterone (1 microM) in the presence or absence of human TNF-alpha (20 ng/ml). Media were removed at 48 and 96 h after culture and progesterone, oestradiol and cAMP in media were measured by radioimmunoassays. FSH stimulated the accumulation of oestradiol from granulosa cells of small follicles during the follicular and luteal phases but not during acyclicity; and TNF reduced oestradiol accumulation in the presence of FSH. Interestingly, in granulosa cells from small follicles, progesterone and cAMP secretion increased in response to FSH and neither was affected by TNF. Thus, TNF specifically inhibited the conversion of testosterone to oestradiol in granulosa cells from small follicles. FSH stimulated oestradiol production by granulosa cells of large follicles obtained only during the follicular phase of the cycle and TNF inhibited the FSH-induced oestradiol secretion. Granulosa cells obtained from large follicles during the luteal phase and during acyclicity did not accumulate oestradiol in response to FSH. However, FSH increased progesterone and cAMP secretion by granulosa cells obtained from large follicles during the follicular and luteal phases. During the luteal phase alone, TNF in combination with FSH increased progesterone accumulation above that of FSH alone. FSH did not increase progesterone, oestradiol or cAMP secretion by granulosa cells obtained from large follicles during acyclicity. Thus, FSH increases progesterone, oestradiol and cAMP secretion by granulosa cells of small follicles during the follicular and luteal phases and TNF appears to inhibit FSH-induced oestradiol secretion specifically in those cells. In large follicles, FSH-stimulated granulosa cell secretion of oestradiol is limited to the follicular phase and this effect can be inhibited by TNF. In addition, when granulosa cells of large follicles do not increase oestradiol secretion in response to FSH, TNF stimulates progesterone secretion.

Tumour necrosis factor-alpha inhibits follicle-stimulating hormone-induced granulosa cell oestradiol secretion in the human: dependence on size of follicle.

This study investigated the effects of tumour necrosis factor-alpha (TNF) on human granulosa cells taken from ovaries of 11 premenopausal women undergoing oophorectomy during the luteal phase of the cycle for reasons unrelated to ovarian pathology. Granulosa cells from follicles ranging from 5-10 mm diameter (small) and from >10-25 mm (large) were subjected to culture for 48 and 96 h. Granulosa cells were cultured with human FSH (1 ng/ml), testosterone (1 microM) and human TNF (10 ng/ml), each alone, and in various combinations. In granulosa cells of small follicles, FSH alone increased progesterone and cAMP accumulation and the conversion of testosterone to oestradiol. In granulosa cells of large follicles, FSH increased progesterone and cAMP accumulation but not the conversion of testosterone to oestradiol. Only in granulosa cells of small follicles did TNF significantly inhibit FSH-induced conversion of testosterone to oestradiol but it was not apparent until the second 48 h of culture and concomitantly TNF did not alter the ability of FSH to stimulate progesterone and cAMP accumulation. In granulosa cells of large follicles, TNF did not alter FSH-stimulated oestradiol, progesterone or cAMP accumulation. Interestingly, progesterone accumulation in the presence of TNF and FSH was significantly greater in granulosa cells of large follicles than in granulosa cells of small follicles. The results indicate that TNF suppresses FSH-induced oestradiol secretion in granulosa cells from small follicles and this modulatory effect of TNF appears to be independent of decreases in progesterone and cAMP. The potential physiological significances of these findings is that TNF may be a relevant cytokine in suppressing FSH-induced oestradiol secretion and follicular growth during the luteal phase of the cycle.

Brooding strategies in solitary ascidians: Corella species from north and south temperate waters

Corella inflata from the northwest Pacific coast of North America and Corella eumyota from southern New Zealand are both ovoviviparous solitary ascidians with very different methods of brood retention. Corella inflata eggs are covered with large follicle cells that are responsible for flotation. The floating eggs are trapped in the upwardly situated atrial chamber, where development ensues. Corella eumyota eggs have small follicle cells and do not float; the follicle cells produce a viscous glue that causes the eggs and embryos to stick to each other and to the right atrium. The follicle cells are not sticky when removed from the ovary but become so in about 15 min. In C. inflata, development to hatching requires 24–26 h at 13 °C and metamorphosis begins 17 h later. Corella eumyota tadpoles hatch at about 25 h at 15 °C and begin metamorphosis 11 h later. Both species retain the tadpoles well beyond hatching and release tadpoles fully competent to metamorphose. In C. inflata the tadpoles swim down and out of the atrium; C. eumyota tadpoles somehow break free of the glue-encrusted vitelline coat and swim out the atrial siphon. Tadpoles of both species swim vigorously when released and then rapidly metamorphose. Most tadpoles metamorphose in 15–20 min. Since both species spawn around dawn, tadpoles are released during the night.

Cellular Localization and Hormonal Regulation of Follicle-Stimulating Hormone and Luteinizing Hormone Receptor Messenger RNAs in the Rat Ovary

The pituitary gonadotropins FSH and LH are key hormones for regulating gametogenesis and steroidogenesis in the ovary and testis. The cell surface receptors that mediate the biological activities of these hormones are thought to be expressed in a cell-specific fashion in the ovary and are regulated as animals progress through the reproductive cycle. Using cloned receptor cDNAs, we have examined the expression and hormonal regulation of the ovarian FSH and LH receptor mRNAs in the rat. A quantitative reverse transcription-polymerase chain reaction amplification scheme was used to measure relative levels of the FSH and LH receptor mRNAs, while in situ hybridization was used to localize FSH and LH receptor transcripts. In immature animals, low levels of FSH receptor mRNA are observed in the granulosa cells of small follicles, while low levels of LH receptor mRNA are found in the thecal cells of these same follicles. After stimulation with PMSG, levels of both mRNAs increase, and the LH receptor mRNA is localized in both the granulosa and thecal cells of large follicles. Further treatment of PMSG-primed animals with hCG results in down-regulation, particularly of the LH receptor mRNA in granulosa cells. In adult animals, LH receptor mRNA levels change dramatically during the estrous cycle, particularly after the preovulatory LH surge. FSH receptor mRNA levels show a similar pattern of change, but the FSH receptor mRNA is of lower abundance and is not as highly regulated as the LH receptor mRNA. FSH receptor mRNA is confined to the granulosa cells of healthy developing follicles, whereas LH receptor mRNA is localized predominantly to thecal cells of small follicles on estrous morning, then appears in the granulosa cells of growing follicles by diestrous morning. LH receptor mRNA is also found in interstitial tissues and corpora lutea throughout much of the estrous cycle. Our results indicate that the gonadotropin receptor genes are regulated in a complex fashion during the recruitment, maturation, and ovulation of the ovarian follicle.

Expression of the Neural Cell Adhesion Molecule in Endocrine Cells of the Ovary*

In the adult mammalian ovary morphogenesis and differentiation processes are under hormonal control and, thus, occur in a highly regulated way during the sexual cycle. Cell-cell interactions, such as cell adhesion and cell separation, are crucial during these events. Here we show that the ovarian endocrine cells, which are prototypes of steroid-producing cells, express neural cell adhesion molecules (NCAMs). The combined use of in situ hybridization histochemistry, immunocytochemistry at the light and electron microscope levels, S1 nuclease protection assays, and Western blotting revealed that in the ovary of the adult rat during the estrus cycle and pregnancy, NCAM mRNA and the 140-kDa isoform of this protein are expressed mainly in granulosa cells of growing preantral and antral follicles and in corpora lutea. Since the granulosa cells lining the forming antrum and the antral fluid were strongly immunoreactive, a role for NCAM in the formation of the follicular antrum is proposed. The expression of NCAM was also associated with luteal cells of the active corpus luteum, indicating a role for NCAM in the morphogenesis of this endocrine compartment. Moreover, thecal cells of large follicles and hypertrophic thecal cells of atretic follicles expressed NCAM, as did interstitial cells, which are derived from thecal cells of atretic follicles. We propose that the adhesion molecule, NCAM, is an important factor involved in the recognition and intercellular interaction of ovarian endocrine cells and, thus, participates in the regulation of the cyclic remodeling processes of the ovarian endocrine compartments.

Transient Expression of Progesterone Receptor Messenger RNA in Ovarian Granuiosa Cells after the Preovulatory Luteinizing Hormone Surge

The ovarian steroid progesterone affects reproductive physiology by regulating the expression of specific genes in target tissues. In an attempt to address the question of whether the ovary itself is a target tissue for progesterone action, we have examined the localization and regulation of progesterone receptor (PR) mRNA in the rat ovary. We used the polymerase chain reaction to clone the steroid-binding domain of the rat PR from uterine cDNA and used this as a probe to isolate a larger cDNA from a rat placental cDNA library. We used RNA filter hybridization, a quantitative reverse transcription-polymerase chain reaction amplification assay, and in situ hybridization to detect PR mRNA in the rat ovary. Expression of the PR gene was initially studied in an immature animal model; 23-day-old rats were treated with either PMSG or PMSG followed by hCG. We found little or no PR mRNA in the ovaries of control or PMSG-treated animals; however, the mRNA was highly expressed in the granulosa cells of large follicles in the ovaries of animals treated with PMSG followed by hCG. Other cell types, including thecal and interstitial cells, did not express detectable levels of PR mRNA. The PR mRNA was induced more than 20-fold in the immature ovary 5 h after hCG administration and was down-regulated to near-basal levels by 12 h after hCG administration. In a subsequent series of experiments, we examined PR gene expression in adult rats during the estrous cycle. The expression of PR mRNA was transient and was tightly coupled to the preovulatory LH surge on proestrous evening. PR mRNA was localized to the granulosa cells of mature ovarian follicles during the estrous cycle. In cycling animals treated with pentobarbital to block the preovulatory LH surge, no induction of PR mRNA on proestrous evening was observed. This transient, hormonally regulated, and cell-specific expression of the PR gene in the rat ovary strongly suggests an important intraovarian function for progesterone during the rat reproductive cycle.

Developmental and hormonal regulation of bovine granulosa cell function in the preovulatory follicle.

Bovine granulosa cells were isolated from small antral, medium antral, and large Graffian follicles (i.e. small, medium, and large preovulatory follicles). Serum-free cultures of granulosa cells were established and found to be viable for 3-6 days of cell culture. Radiolabeled granulosa cell-secreted proteins were obtained and analyzed electrophoretically. No major changes were detected in the protein profiles of small, medium, and large follicle granulosa cells. FSH and insulin, however, had a dramatic effect on granulosa cell-secreted proteins and increased the apparent production of 200K, 65K, 25K, and 15K proteins. The effects of these hormones on the radiolabeled secreted proteins were similar for small, medium, and large follicle granulosa cells. Aromatase activity was high for the first day of serum-free granulosa cell culture and subsequently declined to low levels. Both FSH and insulin alone stimulated aromatase activity, while a combination of hormones resulted in an additive response similar to the stimulation observed with 10% calf serum. Although the level of aromatase activity increased slightly with the size of the follicle, the effects of hormones were independent of follicle size. Progesterone production was low on days 1 and 2 of serum-free granulosa cell culture and high on days 3 and 6 of cell culture. Interestingly, FSH and insulin suppressed progesterone production on day 1 of cell culture for small and medium follicle granulosa cells, but not for large follicle cells. In contrast, hormones stimulated progesterone production on days 3 and 6 of granulosa cell culture, and the level of progesterone production increased with the size of the follicle. The stimulatory effects of hormones on days 3 and 6 of the culture were similar for medium and large follicle granulosa cells, but were altered for small follicle cells. Results indicate that when aromatase activity is high and stimulated by hormones, progesterone levels are low and generally suppressed by the same regulatory agents. Conversely when progesterone levels are high and hormone responsive, aromatase activity is low. The inverse relationship between aromatase activity and progesterone production implies that bovine granulosa cells alter their differentiated state in culture from an estrogen-producing cell to a progesterone-producing cell. Combined observations indicate that the results obtained on day 1 of culture probably reflect the developmental and hormonal regulation of granulosa cell function in the preovulatory follicle, while data obtained at later times in culture reflect the ability of the cell to synthesize progesterone and develop a luteinization-like activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Secretion of follicle-regulatory protein by porcine granulosa cells.

Follicle-regulatory protein (FRP) affects ovarian steroidogenesis and thus follicular maturation. However, secretion of FRP by cells from different-sized follicles as well as the modulation of FRP production by gonadotropins and locally produced steroids are unknown. To evaluate which cell type secretes FRP, theca and granulosa cells were obtained from porcine follicles. In addition, the effects of follicle-stimulating hormone (FSH) and steroids on FRP secretion from granulosa cells of small (less than 3 mm), medium (3-6 mm), and large (greater than 8 mm) porcine follicles and theca cells of large follicles were determined. Granulosa cells were obtained from follicular aspirates, whereas theca cells were recovered after digestion of the stereomicroscopically removed thecal layer. Both were cultured in monolayer in serum-free medium. Granulosa cells were treated as follows: 1) control; 2) FSH (250 ng/ml); 3) progesterone (500 ng/ml, 3 micrograms/ml), or estradiol-17 beta (500 ng/ml, 4 micrograms/ml), or dihydrotestosterone (500 ng/ml, 1 microgram/ml); 4) FSH + progesterone, or estradiol-17 beta, or dihydrotestosterone. Theca cells received the same treatment except that human chorionic gonadotropin (hCG) (5m IU/ml) was used in place of FSH. At 48 or 96 h, media were removed and FRP was quantitated by an Enzyme-Linked Immunosorbent Assay (ELISA). FRP was identified in granulosal medium from follicles of all sizes, but was not present in thecal cultures. At 48 h, granulosa cells from small and medium-sized follicles produced more FRP (20.04 +/- 4.4, 35.42 +/- 4.1 immunoreactive units [IRU]) than cells from large (3.53 +/- 0.97 IRU) follicles.(ABSTRACT TRUNCATED AT 250 WORDS)

Ovarian follicular growth, function and turnover in cattle: a review.

Studies in cattle assessing changes in number and size of antral follicles, concentrations of estradiol, androgens and progesterone in serum and follicular fluid, and numbers of gonadotropin receptors per follicle during repetitive estrous cycles and postpartum anestrus are reviewed. The rate of growth of small follicles (1 to 3 mm) into larger follicles increases as the estrous cycle progresses from d 1 to 18 (d 0 = estrus). Size of the largest antral follicle present on the ovary also increases with advancement of the estrous cycle. Most large follicles (greater than 10 mm) persist on the ovarian surface for 5 d or more between d 3 and 13 of the bovine estrous cycle. After d 13, most of these large follicles are replaced more frequently by new growing follicles (turnover) with an increased probability for recruitment of the ovulatory follicle after d 18. More research is needed to determine the time required for growth of bovine follicles from small to large antral size and evoke recruitment of the ovulatory follicle. Factors that regulate selection of the ovulatory follicle are unknown but may involve increased frequency of LH pulses in blood, altered blood flow and(or) changes in intrafollicular steroids and proteins. Quantitative evaluation of ovarian follicles indicated occurrence of consistent short-term changes in fluid estradiol and numbers of luteinizing hormone receptors in cells of large follicles only during the pre-ovulatory period. Presumably, low concentrations of follicular estradiol found during most of the estrous cycle are not due to a lack of aromatizable precursor or follicle-stimulating hormone receptors. Follicular fluid concentrations of progesterone increase only near the time of ovulation. Little is known about changes in follicular growth, turnover and function during postpartum anestrus in cattle. However, preliminary data suggest that the steroidogenic capacity of large follicles changes markedly during the postpartum period.

Internalization of receptor-bound human chorionic gonadotrophin in preovulatory rat granulosa cells in vivo.

The subcellular distribution of 125I-labelled human chorionic gonadotrophin (hCG) in preovulatory rat granulosa cells was studied in vivo. Pregnant mare serum gonadotrophin-pretreated immature female rats received an iv injection of [125I]hCG a few hours before the endogenous preovulatory gonadotrophin surge. The animals were killed at 2 or 6 h after the [125I]hCG injections. Light microscope autoradiographs showed that the mural granulosa cells of large follicles were the most highly labelled cells in the ovaries. Electron microscope autoradiography was used to study the subcellular distribution of radioactivity in the mural granulosa cells. At 2 h 45% of the counted silver grains were associated with the plasma membrane and 10% with the lysosomes, at 6 h the values were 51% and 9%, respectively. The distribution of the observed silver grains was compared with the generated expected source to grain pairs by computerized linear multiple regression analysis. The magnitudes of the regression coefficients revealed that the plasma membrane and the lysosomes were the only specifically 125I-labelled organelles, that a few radioactive molecules were located diffusely over the cytoplasm at 2 h and that the 125I-radioactivity of the nuclei was negligible. The present results suggest that preovulatory rat granulosa cells are in vivo able to internalize into lysosomes [125I]hCG initially bound to LH/hCG receptors of the plasma membrane.

Estrogen secretion and gonadotropin binding by individual bovine follicles during estrus.

This study was conducted to examine the capacity of individual bovine follicles to secrete estradiol at estrus and to determine if the rate of estrogen secretion can be altered by the level of feed intake. Estrus was synchronized in 16 mature beef cows after 80 d on a high or low feeding regimen (130 or 65% of NRC requirements for total digestible nutrients). Ovaries were removed either 2 or 16 h after the second behavioral estrus following synchronization. To define the preovulatory luteinizing hormone (LH) surge, blood samples were taken at frequent intervals as estrus approached. The nine largest follicles (per cow) were isolated from the ovaries, incubated individually in medium 199 (M 199) for 4 h, and the medium then assayed for estradiol-17β (E2). Testosterone (250 ng/ml) was added to the incubation medium of one-half of the follicles for the last 2 h incubation. After incubation, binding of iodinated gonadotropins to the theca and granulosa cells of the largest follicle was quantitated. The LH profile for each cow was determined, and the time of ovary removal classified as occurring on the ascending portion of the LH peak or after (postpeak) the peak returned to baseline. The largest follicle of each cow secreted E2 at a rate two to three orders of magnitude greater than the smaller follicles. Secretion of E2 was much greater by the largest follicle in the peak group than in the postpeak group (51.6 vs 8.2 ng•ml−1• 30 min−1). Addition of testosterone to the M 199 had no effect on E2 secretion by either the largest or small follicles. Binding of human chorionic gonadotropin (hCG) to theca cells of large follicles was correlated with E2 secretion (r = .68, P<.01) and with basal LH concentrations (r = .59, P<.05) when computed across all treatment groups. When calculated within the peak and postpeak groups, these correlations were not significant. There was no effect of nutrition on E2 secretion (34.7 vs 32.9 ng•ml−1•30 min−1), serum concentration of LH at the preovulatory surge (43.4 vs 42.1 ng/ml) or serum baseline concentration of LH (2.6 vs 2.9 ng/ml); all for high vs low feed, respectively. These results indicate that the largest follicle is responsible for most of the E2 secretion of the ovary pair at estrus and that the E2 secretion by the largest follicle decreases rapidly at the time of the LH peak.

An ultrastructural study of oogenesis inStreblospio benedicti (spionidae), with remarks on diversity of vitellogenic mechanisms in polychaeta

The ultrastructural features of oogenesis were examined in the spionid polychaeteStreblospio benedicti. Paired ovaries are attached to the genital blood vessels extending into the coelomic space from the circumintestinal sinus. The genital blood vessel wall is composed of flattened, peritoneal cells, large follicle cells and developing oocytes. Vitellogenesis occurs while the oocytes are attached to the blood vessel wall. Two morphologically distinguishable types of yolk are synthesized. Type I is synthesized first by an autosynthetic process apparently involving pinocytosis and the conjoined efforts of the Golgi complex and rough endoplasmic reticulum. Type II yolk appears later through a heterosynthetic process involving the infolding of the oolemma and the sequestering of materials from the blood vessel lumen by endocytosis. During this process, blood pigment molecules appear to be incorporated into endocytotic pits, vesicles and eventually the forming yolk body. The significance of heterosynthetic yolk formation to the general reproductive strategies of polychaetous annelids is discussed.

The differentiation and proliferation of follicle cells during oocyte growth in Lacerta sicula

The follicular epithelium of the lizard oocytes undergoes structural and morphological modifications throughout oocyte growth. During this process the number of follicle cells increases and the epithelium acquires a multilayered and polymorphic organization which is characterized by the appearance of large follicle cells (intermediate and pyriform cells). The number of large cells also increases during oocyte growth and this increase parallels that of small cells. However, only the small cells become labelled one hour after [3H-]thymidine administration. Large cells have been found labelled after a longer period of time, i.e. 4--5 months after isotope injection. All these results together indicate that large follicle cells arise from the differentiation of small cells.

Adenylate Cyclase of Porcine Granulosa Cells: Differential Response to Gonadotropins during Follicle Maturation*

The human FSH (hFSH)- and human LH (hLH)/hCG-mediated adenylate cyclase activities of granulosa cells from small (1–2 mm), medium (3–5 mm), and large (6–12 mm) porcine ovarian follicles were studied. The adenylate cyclase of small follicle cells was highly sensitive to hFSH with an 8- to 13-fold increase over the basal activity, whereas hCG had little effect (only a 2-fold increase) on the enzyme activity. The stimulation by hFSH cannot be accounted for by hLH contamination, since hFSH had a larger stimulatory effect and a lower apparent concentration for half-maximal stimulation (apparent Km, 370 ng/ml) than that of hLH (apparent Km, 1700 ng/ml). The adenylate cyclase of granulosa cells became increasingly responsive to hCG and hLH as the follicles developed. hCG at the saturating concentration stimulated the enzyme activity with a 4-fold increase in medium follicle cells and with a 10- to 14-fold increase in large follicle cells without change in the apparent Km. hLH had a stimulatory effect similar to ...

MATURATIONAL CHANGES IN SHEEP OVARIAN FOLLICLES: GONADOTROPHIC STIMULATION OF CYCLIC AMP PRODUCTION BY ISOLATED THECA AND GRANULOSA CELLS

Theca and granulosa tissues isolated from sheep ovarian follicles of different sizes were incubated in the presence of human chorionic gonadotrophin (HCG; 5 IU/ml) or follicle stimulating hormone (FSH; 5 microgram NIH-FSH-S11/ml) for 40 min. Changes in the total amounts of cyclic 3',5'-adenosine monophosphate (cAMP) were used as an index of the responsiveness of these preparations to the hormones. Thecal tissue of both large (4-6 mm in diameter) and small (1-3 mm) follicles responded similarly to gonadotrophins. Granulosa cells from small follicles failed to respond to stimulation by HCG. FSH, however, consistently increased cAMP production in comparison with controls or cells treated with HCG. Granulosa cells of large follicles responded to both HCG and FSH.

The Porcine Ovarian Follicle. II. Electron Microscopic Study of Surface Features of Granulosa Cells at Different Stages of Development1

Surface features of granulosa cells from porcine ovarian follicles were studied at different stages of development by electron microscopy. The presence ofnumerous microvilli on the luminal surface granulosa cells of large follicles and their absence on granulosa cells from small and medium follicles were observed. Microfilaments within the microvilli were also observed. A possible relationship between these structural features and the appearance of trophic hormone receptors is discussed.

The Porcine Ovarian Follicle: III. Development of Chorionic Gonadotropin Receptors Associated with Increase in Adenyl Cyclase Activity During Follicle Maturation

Specific binding of human chorionic gonadotropin (hCG), and the hCG-sensitive adenyl cyclase of granulosa cells from small (1-2 mm), medium (3-5 mm), and large (6-12 mm) porcine ovarian follicles have been studied. The number of hCG-binding sites per cell (n) increases during follicle maturation without a change in the binding affinity. The values for n were 300-400 for small, 1,000-1,600 for medium, and 8,200-10,000 for large-follicle cells. The dissociation constant is 2.4 X 10(-10)M for all cells. hCG-sensitive adenyl cyclase was demonstrated in porcine granulosa cells. The adenyl cyclase system of granulosa cells becomes increasingly responsive to hCG stimulation during follicle development. Maximal adenyl cyclase activation by hCG (1 mug/ml) was 240, 750, and 7,000 molecules of cyclic AMP formed/sec/cell, respectively, for small, medium, and large follicle cell. The concentration of hCG giving half-maximal stimulation (1.0 X 10(-9)M) was similar for both large and medium follicle cells. It is concluded that: 1) an increase in hCG receptor sites per cell occurs during maturation of the porcine ovarian follicle without change of binding affinity, and 2) the increase in the number of hCG receptors correlates well with hCG-sensitive adenyl cyclase activity during follicle development.