Influence of luteinizing hormone and progesterone on maturation and fertilizability of rat oocytes in vitro

The effects of luteinizing hormone (NIH-bovine LH) and progesterone on maturation in vitro of oocyte-cumulus complexes from adult proestrous rats were studied by comparing proportions of oocytes showing germinal vesicle breakdown, mucification of the cumulus oophorus, and fertilizability. Addition of either or both of the hormones to the medium in concentrations between 1.25 and 10 μg/ml during maturation had no discernible effect on germinal vesicle breakdown or on fertilization. Mucification was stimulated by LH and even more by LH plus progesterone. It was concluded that maturation in vivo is the result of concerted action of the two hormones. However, addition of LH + progesterone had no effect on the fertilizability of these oocytes. We attribute this to a relative insensitivity of the system for fertilization in vitro to subtle changes in the oocyte.     

Induction of maturation in follicle-enclosed oocytes: the response to gonadotropins at different stages of follicular development

Antral follicles, isolated from either nontreated or pregnant mare's serum gonadotropin (PMSG)-primed 27-day-old rats, were incubated in the absence or the presence of either luteinizing hormone (LH), follicle-stimulating hormone (FSH), or forskolin. The effect of these agents on oocyte maturation and cyclic adenosine 3',5'-monophosphate (cAMP) accumulation was studied and compared. Both gonadotropins, LH and FSH, as well as forskolin, effectively induced maturation of oocytes enclosed by large antral follicles isolated from PMSG-primed rats. On the other hand, we found that maturation of oocytes enclosed by small antral follicles, isolated from nonprimed and PMSG-primed rats, could be induced by either FSH or forskolin but not by LH. cAMP determinations revealed that, in spite of the inability of LH to induce oocyte maturation, elevated concentrations of the nucleotide were detectable in small antral follicles exposed to this gonadotropin. Since granulosa cells isolated from the large but not the small antral follicles were stimulated by LH to generate cAMP, the elevation of cAMP concentrations in the small antral follicle apparently represented the response of the theca cells to this gonadotropin. Since it is the ability of the granulosa cells to interact with the hormone that determines whether or not oocyte maturation will occur, we suggest that the granulosa, but not the theca cells, mediate LH action to induce oocyte maturation.     

In the absence of protein, estradiol suppresses meiosis of porcine oocytes in vitro

The effect of estradiol on the spontaneous maturation of porcine oocytes was investigated. Cumulus-enclosed (intact) and cumulus-free (denuded) oocytes were cultured in the presence of estradiol-17 beta (0 to 10 microgram/ml) in a chemically defined bicarbonate-buffered medium that contained either dextran or BSA, or in a complex Hepes-buffered medium that was supplemented with serum. After 24 hr, chromatin spreads were prepared and meiotic maturation was scored. The biochemical integrities of the cumulus cells were assessed by determination of the estradiol and progesterone content of spent media after culture of intact oocytes in the presence of 0.5 X 10(-6) M testosterone and 10 microgram/ml follicle-stimulating hormone. Estradiol did not significantly affect the onset of maturation of either intact or denuded oocytes that were cultured in medium containing either BSA or serum. In serum-supplemented medium, however, the progression of maturation beyond metaphase I was significantly affected by the steroid in a dose-dependent manner. The steroid significantly inhibited the release from meiotic arrest of both types of oocyte cultured in medium supplemented with dextran. Supplementation of all media with testosterone and FSH significantly stimulated the synthesis of estradiol by the cumulus cells, compared with that of control groups. The synthesis of progesterone, however, was significantly stimulated by testosterone and FSH only in the BSA and serum-supplemented media. It is concluded that exogenous estradiol has the capacity to arrest meiosis in vitro but that this capacity can only be expressed if no exogenous protein(s) is present. In the absence of exogenous protein, progesterone synthesis by the adherent cumulus cells is minimal.     

Luteinizing hormone receptor formation in cumulus cells surrounding porcine oocytes and its role during meiotic maturation of porcine oocytes

We investigated the formation of LH receptor (LHR) in cumulus cells surrounding porcine oocytes and the role of LHR in meiotic maturation of oocytes. At least three splice variants of LHR mRNA were detected in cumulus cells, in addition to the full-length form. Low levels of three types of products were seen in cumulus cells from cumulus oocytes complexes (COCs), whereas the full-length form was significantly increased by 12-h cultivation with FSH. The addition of FSH also significantly increased the binding level of biotinylated hCG to COCs. The formation of LHR in FSH-stimulated cumulus cells was not affected by additional 0.5 mM phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX), and the oocytes were synchronized to the germinal vesicle (GV) II stage by exposure to 0.5 mM IBMX and FSH for 20 h. The binding of LH to its receptor induced a further increase in cAMP level and progesterone production and acceleration of meiotic progression to the metaphase I stage. The oocytes cultured with LH for 24 h following cultivation with FSH and IBMX were used for in vitro fertilization. At 6 days after in vitro fertilization, blastocyst rate in oocytes matured under these conditions was significantly higher than that of oocytes cultured in the absence of LH. Treatment of oocytes with FSH and 0.5 mM IBMX to express LH receptor in cumulus cells while holding oocytes at the GV II stage is a very beneficial way to produce in vitro-matured oocytes, which have high developmental competence.     

Effects of LH and FSH on the maturation of pig oocytes in vitro

This research was designed to investigate the effects of LH and FSH (50 ng/ml) on pig oocyte maturation in vitro. The following parameters were studied: a) the degree of heterologous coupling between cumulus cells and oocytes, evaluated by measuring the (3)H-uridine and (3)H-choline uptake in cumulus enclosed oocytes; b) meiotic maturation; c) cytoplasmatic maturation, evaluated by analyzing the ability of the oocytes to promote male pronucleus formation after in vitro fertilization. Despite the marked cumuli expansion induced by gonadotropins, uridine uptake was not influenced by LH or FSH. By contrast, choline uptake in LH-treated oocytes was significantly higher than in FSH-treated or control oocytes (3199 cpm +/- 251 vs 1686 cpm +/- 142, P<0.01). Gonadotropins accelerated meiotic progression, and after 30 hours of culture the percentage of oocytes at the germinal vesicle stage was significantly lower (P<0.01) in LH-(24%, 24 102 ) and FSH-(20%, 18 90 ) treated oocytes than in control oocytes (76%, 64 84 ). After 44 hours of culture, the percentage of oocytes reaching the MII stage was significantly higher (P<0.01) in the presence of LH (76%, 92 120 ) and FSH (86%, 92 108 ) than in the controls (35%, 40 116 ). The percentage of oocytes capable of sustaining male pronucleus formation was similar in the control (48.4%, 63 132 ) and FSH-treated oocytes (44.3%, 51 116 ), while it was markedly increased (P<0.01) by the addition of LH (72.7%, 143 197 ). The data reported indicate that in vitro pig oocytes tend to undergo meiotic maturation even in the absence of hormones. However, in our in vitro system, LH and FSH accelerated and facilitated meiotic progression, and LH selectively improved cytoplasmic maturation which is required to promote the formation of a male pronucleus.     

Effects of gonadotropins and steroids on the subsequent fertilizability of extrafollicular bovine oocytes cultured in vitro

Effects of gonadotropins (2 i.u./ml follicle stimulating hormone, FSH and 10 μg/ml luteinizing hormone, LH) and steroids (1 μg/ml oestradiol, E and progesterone, P) on the fertilizability of extrafollicular bovine oocytes cultured in vitro and transferred in either the rabbit oviduct (Experiment I) or glass test tubes (Experiment II) were investigated. Bovine oocytes collected from follicles of 2–5 mm in diameter were cultured in vitro for 27 h in a medium containing Ham's F-12, 20% (v/v) bovine fetal serum and antibiotics. The combination of the hormones added to the medium was as follows; (1) none (control), (2) E, (3) LH, (4) LH + E, (5) FSH + LH + E, and (6) FSH + LH + E + P. All oocytes were recovered 24 h after insemination and examined for the presence of the pronuclei and a sperm tail with the midpiece in the oocyte cytoplasm, and the extrusion of the second polar body.In Experiment I, 630 of 704 transferred oocytes (85.7%) were recovered from the rabbit oviduct. The maturation rates of these oocytes (overall 61.1%) were not significantly affected by gonadotropins and steroids. Of the 741 of 920 oocytes recovered from test tubes in Experiment II, the maturation rates of them (overall 64.2%) were significantly increased (P < 0.05) by addition of LH (72.9%) and FSH + LH + E + P (74.1%) as compared with controls (55.4%). Fertilization rates were increased (P < 0.05) by the addition of FSH + LH + E compared with the controls in both Experiments I (31.1% and 14.0%) and II (36.2% and 20.8%). Furthermore, the proportion of fertilized eggs in hormone treated groups was the highest in each experiment. The present study indicates that the addition of FSH, LH and E to a medium has improved the fertilizability of extrafollicular bovine oocytes cultured in vitro.     

Suppression of spontaneous maturation of isolated cumulus-free mouse oocytes by a calmodulin antagonist

Oocytes isolated from antral follicles undergo spontaneous maturation when cultured in vitro. W7, a calmodulin antagonist, at concentration of more than 50 microM blocked the occurrence of spontaneous germinal vesicle breakdown (GVBD) in isolated cumulus-free mouse oocytes. The inhibition of maturation was observed in more than 90% of oocytes when W7 was added within 15 min after the initiation of incubation of the oocytes. The block was partially reversible. Hypoxanthine, estradiol-17 beta, testosterone and progesterone did not influence the inhibition induced with W7. The present results suggest that calmodulin is involved in the early stage of mouse oocyte maturation.     

Short time priming of pig cumulus-oocyte complexes with FSH and forskolin in the presence of hypoxanthine stimulates cumulus cells to secrete a meiosis-activating substance

This study evaluated the effect of forskolin and FSH on pig oocyte maturation when cultured in a maturation inhibiting system. Ovaries from prepubertal gilts were collected at a local slaughterhouse. Oocytes were cultured in a hypoxanthine (HX 4 mM) containing M 199 for 24 or 40 h with or without forskolin and FSH treatment. After the culture, we examined germinal vesicle breakdown (GVBD) and polar body (PB) formation. Two experiments were designed. (1) Cumulus enclosed oocytes (CEO) were cultured for 24 or 40 h with or without different doses of forskolin and FSH. (2) CEO were primed by forskolin and FSH for different times and then transferred into an HX-medium for a further culture. The total culture period was 24 h. The results revealed that 4 mM HX markedly prevented pig CEO from undergoing GVBD. After 24 and 40 h culture, FSH (50-200 U/L) stimulated oocytes to resume meiosis by overcoming the inhibition of HX. Both GVBD and PB formation were increased (P < 0.002 and 0.01 respectively) after 40 h exposed to FSH. Forskolin showed a biphasic effect on CEO maturation. Within 24 h forskolin, in combination with HX, inhibited oocytes maturation. The GVBD percentage was significantly decreased compared to HX alone group (2% to 20%, P < 0.01), whereas no inhibition was observed after 40 h of culture. The second experiment showed that forskolin (3 microM) and FSH (100 U/L) priming CEO could time-dependently induce oocyte maturation by overriding the inhibition of HX. After 30 and 60 min priming by FSH or forskolin, the GVBD and PB percentage was significantly increased (P < 0.002 and 0.01 respectively). No difference of GVBD percentage was observed between FSH short time priming group and FSH long time presentation group. In conclusion, we found that forskolin and FSH in vitro can stimulate pig cumulus cells to secrete a meiosis-activating substance which induces the oocyte to overcome the inhibition of hypoxanthine and undergo GVBD.     

Effects of follicle-stimulating hormone and ovarian steroids during vitro meiotic maturation on fertilization of rat oocytes

The present study examined the effects of gonadotropins and ovarian steroids during in vitro meiotic maturation of rat oocytes on their ability to undergo in vitro fertilization. Fully grown oocytes were isolated from antral follicles of immature rats and cultured as oocyte-cumulus cell complexes (OCC) under conditions in which completion of meiotic maturation occurs spontaneously. They were then exposed to spermatozoa under conditions in which oocytes matured in vivo exhibit high fertilization rates. Compared with oocytes from pregnant mare's serum gonadotropin (PMSG) or follicle-stimulating hormone (FSH)-treated rats, a simiiar proportion of the oocytes (>80%) from untreated rats underwent germinal vesicle breakdown, but such oocytes had a lower rate of fertilization (70% vs. 20%). The presence of FSH during in vitro maturation restored the fertilization rate for oocytes from untreated rats, while a cytochrome P450 inhibitor, aminoglutethimide phosphate abolished this beneficial effect of FSH. The addition of progesterone during the in vitro maturation period duplicated the beneficial effect of FSH on fertilization rate of oocytes from untreated rats; oestradiol-17β was less effective in this regard, and 5α-dihydrotestosterone was ineffective. These findings indicate that FSH and progesterone, although having no apparent effect on nuclear maturation of the oocyte, play an important role during oocyte maturation in enabling normal fertilization to occur.     

Developmental potential of rabbit oocyte matured in vitro: the possible contribution of prolactin

The present study was undertaken to define hormonal conditions for in vitro maturation that support subsequent fertilization and embryonic development. Follicular oocytes were recovered from nonstimulated rabbit ovaries and cultured for 12 h in Brackett's medium supplemented with or without hormones. Matured oocytes were inseminated in vitro and transferred 12 h later to Ham's F-10 medium supplemented with 20% fetal calf serum. The initial cleavage frequency of matured oocytes in Brackett's medium was comparable to the frequency of development for in vitro-matured oocytes under various hormonal conditions. However, the addition of estradiol (E2, 1 microgram/ml) to incubation medium containing luteinizing hormone (LH) and follicle-stimulating hormone (FSH) increased significantly (p less than 0.001) the percentage of embryos achieving morula or blastocyst formation (16/98, 16.3%), as compared to the mature oocytes in medium containing LH, LH plus FSH, or no hormone. The addition of prolactin (PRL) to the maturation medium increased the percentage of development to organized embryos in a dose-dependent manner. In vitro-matured oocytes in medium containing LH, FSH, and PRL exhibited a significantly (p less than 0.001) lower incidence of developmental competence (5/95, 5.3%) than oocytes matured in the presence of E2 in conjunction with pituitary hormones (43/89, 48.3%). These results demonstrate that hormonal composition in the environment of the oocyte is critical for acquisition of developmental capacity. PRL as well as E2 appears to be an important constituent in the process of oocyte maturation, promoting preimplantation embryonic development.     

In vitro fertilization and development of bovine oocytes matured in serum-free medium

This study was undertaken to investigate the effects of supplementation of serum (fetal calf serum), gonadotropins (LH, FSH, prolactin) and estradiol-17 beta (E2) to culture medium during in vitro maturation of bovine cumulus oocyte complexes on subsequent fertilization and development to the blastocyst stage in vitro. Serum supplementation during bovine oocyte maturation was not required but hormonal supplementation, gonadotropins (LH + FSH) and E2, enhanced the fertilizability and developmental ability of bovine oocytes matured in vitro. The addition of prolactin to maturation medium containing LH, FSH, and E2 did not further enhance frequencies of fertilization and development.     

Stimulation and inhibition of rat oocyte meiosis by forskolin

The adenylate cyclase activator forskolin was used to study the role of cAMP for oocyte meiosis and follicular steroid secretion. Follicular and cumulus cAMP production was stimulated dose-dependently by forskolin, as was the follicular secretion of progesterone, testosterone and estradiol. Forskolin induced meiosis in follicle-enclosed oocytes with a maximal effect at 1 microM, with lower and higher concentrations being less effective. The spontaneous resumption of meiosis in isolated cumulus-enclosed oocytes was dose-dependently retarded by forskolin. Meiosis of cumulus-free oocytes was also retarded but only slightly. These data support the earlier hypothesis that a limited increase in follicular cAMP levels triggers meiosis, whereas sustained levels of cAMP in the oocyte itself prevent meiosis.     

Dichotomous effects of forskolin on somatic and germ cell components of the ovarian follicle: evidence of cAMP involvement in steroid production and action

The role of cyclic AMP (cAMP) in ovarian follicular functions in Rana pipiens was investigated with the use of the adenylate cyclase stimulator, forskolin, which is thought to elevate intracellular level of cAMP. Effects of forskolin on oocyte germinal vesicle breakdown (GVBD) and on progesterone production by the follicles were assessed during the course of in vitro culture. Addition of forskolin to culture medium suppressed both progesterone-and frog pituitary homogenate (FPH)-induced meiotic maturation of the oocytes. Inhibitory effects of forskolin were essentially reversible and forskolin completely inhibited GVBD when added during the first four hours of incubation following exposure to progesterone. Forskolin alone stimulated a low level progesterone production by isolated follicles, but markedly stimulated progesterone production when it was supplemented with a low dose of FPH (0.005 pituitary equivalent/ml). Thus, forskolin acts synergistically with FPH on follicle cells to stimulate progesterone production. A higher dose of FPH (0.05 pitui. eq./ml) produced no additional synergistic effect of forskolin. Therefore, forskolin appears to have two contradictory functions in ovarian follicles: it augments FPH induced follicle secretion of meiosis initiator, progesterone, and simultaneously suppresses the maturation of the oocytes triggered by exogenous progesterone or FPH. The data presented indicate that there are two independent adenylate cyclase systems in the ovarian follicles which have separate functions: one in the follicle cells and the other in the oocyte. The two enzyme systems are thus compartmentalized and regulate different biological functions using the same messenger, cAMP. The data provide evidence that in amphibians, as in mammals, pituitary hormones regulate steroid hormone production by follicle cells via a cyclic AMP system. Thus, control of oocyte maturation induction appears to be determined by the relative levels of cAMP present in the follicle cells and oocytes.     

Induction of G protein-coupled estrogen receptor (GPER) and nuclear steroid hormone receptors by gonadotropins in human granulosa cells

Estradiol and progesterone mediate their actions by binding to classical nuclear receptors, estrogen receptor α (ERα) and estrogen receptor β (ERβ) and progesterone receptor A and B (PR-A and PR-B) and the non-classical G protein-coupled estrogen receptor (GPER). Several animal knock-out models have shown the importance of the receptors for growth of the oocyte and ovulation. The aim of our study was to identify GPER in human granulosa cells (GC) for the first time. Moreover, the effect of different doses of gonadotropins on estrogen and progesterone receptors in the human ovary should be investigated as follicle stimulating hormone (FSH) and luteinizing hormone (LH) are also responsible for numerous mechanisms in the ovary like induction of the steroid biosynthesis. Human GC were cultured in vitro and stimulated with different doses of recombinant human FSH or LH. Receptor expression was analyzed by immunocytochemistry and quantitative real-time RT-PCR. GPER could be identified for the first time in human GC. It could be shown that high concentrations of LH increase GPER protein expression. Furthermore FSH and LH increased ERβ, PR-A and PR-B significantly on protein level. These findings were verified for high doses of FSH and LH on mRNA level. ERα was not affected with FSH or LH. We assume that gonadotropins induce GPER, ERβ and PR in luteinized granulosa cells.     

Failure of gonadotropins to induce in vitro maturation of mouse oocytes treated with dibutyryl cyclic AMP.

In sufficient concentration, dibutyryl cAMP (DBC) prevents the spontaneous in vitro maturation of mouse oocytes. The effects of luteinizing hormone (LH) and follicle stimulating hormone (FSH) on this inhibition were tested in an oil-free chamber-slide culture system. Mouse oocytes devoid of cumulus cells were incubated in the presence of DBC and/or gonadotropins. Oocytes cultured with follicle cells were similarly treated. Whether follicle cells were present or absent, DBC (100 or 500 mug/ml) prevented germinal vesicle breakdown in more than 95% of the oocytes cultured. Neither LH nor FSH in a wide range of concentrations acted directly on the oocytes or indirectly through the follicle cells to initiate maturation in oocytes incubated with 100 mug DBC/ml. The combination of LH (5 mug/ml) and FSH (10 mug/ml) was also ineffective in overcoming the block induced by either 100 or 50 mug DBC/ml. Maturation of oocytes in each of the DBC-free LH or FSH treatments was comparable to that occurring in control medium which did not contain exogenous gonadotropins or DBC. It was concluded that cultured oocytes treated with DBC are not a satisfactory model for studying the steps by which gonadotropins trigger the resumption of meiosis in mammalian oocytes.     

Effects of epidermal growth factor and hormones on granulosa expansion and nuclear maturation of dog oocytes in vitro

Gonadotropins, steroids and growth factors stimulate or inhibit cumulus expansion, nuclear maturation, or both, of most mammalian oocytes in vitro. The objective was to evaluate the effects of epidermal growth factor (EGF) and various hormone combinations on in vitro granulosa/cumulus (G-C) expansion and nuclear maturation of domestic dog oocytes derived from advanced preantral and early antral follicles. Follicles were collected after enzymatic digestion of ovarian tissue and cultured for 66 h in F-12/DME with 20% fetal bovine serum, 2mM glutamine and 1% antibiotic-antimycotic (Control). Treatments comprised the following groups; each was cultured both with and without EGF (5 ng/mL): Control, FSH (0.5 microg/mL), LH (5 microg/mL), estradiol-17beta (E2, 1 microg/mL), FSH+LH, and FSH+LH+E2. Granulosa/cumulus expansion was scored on a scale of 0 (no expansion) to +3 (maximum expansion). The interaction between EGF and hormone treatment affected (P=0.011) maximum G-C expansion. With the exception of the E2 group, EGF increased (P<0.05) the proportion of oocytes exhibiting +3 expansion. The synergism of E2 with FSH+LH enhanced maximum G-C expansion; compared to all other treatments, the greatest expansion was observed in the FSH+LH+E2+EGF group (83.5+/-3.5%). When cultured in EGF alone, oocytes failed to reach metaphase I-II (MI-MII) stages. The interaction between EGF and hormone treatment tended (P=0.089) to increase the proportion of oocytes resuming or completing nuclear maturation (GVBD-MII). In addition, supplementing culture media with hormones increased (P=0.010) the GVBD-MII rate. Therefore, EGF in combination with FSH and LH enhanced G-C expansion of cultured canine oocytes, with no significant effect on the proportion of oocytes derived from advanced preantral and early antral follicles that reached MI-MII.     

Spontaneous maturation in vitro of cumulus-enclosed rat oocytes is inhibited by forskolin

We have recently reported that the adenylate cyclase activator, forskolin, induces in the rat ovarian follicle both cAMP accumulation and oocyte maturation. We demonstrate here, on the other hand, that the spontaneous maturation in vitro of isolated rat cumulus-enclosed oocytes is inhibited by forskolin. The inhibitory effect of forskolin is dose dependent with an ED50 at 15 microM. Forskolin inhibition decreases gradually with time, being completely relieved by 20 h of culture. Methylisobutylxanthine significantly prolongs the duration of the inhibitory action of forskolin. In addition to its inhibitory effect on oocyte maturation, forskolin triggers the cumulus-oocyte complex to generate cAMP. Cyclic AMP accumulation is maximally stimulated by 100 microM of forskolin with an ED50 at 60 microM. The potency of the cumulus-oocyte complex to respond to forskolin in terms of cAMP accumulation decreases with time. The pattern of the decrease in the potency of the cumulus-oocyte complexes to generate cAMP corresponds with the relief of its inhibitory influence on the oocyte. These results indicate that inhibition of maturation of the cumulus-enclosed oocyte may be coupled to elevation of cAMP levels in the cumulus-oocyte complex. As isolated cumulus-free oocytes are not inhibited by forskolin, we suggest that in the cumulus-enclosed oocyte system, cAMP generated by the cumulus cells is apparently transferred to the oocyte and maintains it in a meiotically arrested state. Maturation in this system occurs upon relief of inhibition which results from cessation of cAMP generation by the cumulus cells.     

Effect of cyanoketone on follicle-stimulating hormone (FSH) induction of receptors for FSH in granulosa cells of the rat

Follicle-stimulating hormone (FSH) enhances the conversion of testosterone or androstenedione into estradiol by stimulating the aromatase enzyme system. Estradiol also enhances FSH action. Thus, a synergistic action of FSH and estradiol may be required for maturation of ovarian follicles. We hypothesized that estradiol may be required for FSH action. Thus, blocking estrogen synthesis should prevent FSH-induced increases in FSH receptors. Hypophysectomized rats were divided into five groups and injected subcutaneously with: 1) saline, 2) cyanoketone (0.05 mg, blocks the conversion of pregnenolone to progesterone), 3) ovine FSH (oFSH, 200 micrograms), 4) cyanoketone then oFSH 24 h later, or 5) cyanoketone plus estradiol [or progesterone, testosterone, promegestrone (R5020), dihydrotestosterone (DHT), 2 mg], then FSH 24 h later. Animals were decapitated at 0, 12 or 24 h after an injection of oFSH, and membrane receptors for FSH and luteinizing hormone (LH), plus nuclear receptors for estradiol from granulosa cells, were measured. LH receptor levels were increased only after administration of FSH and estradiol. At 0 and 24 h, numbers of FSH or estradiol receptors were similar in saline- and cyanoketone-treated animals. FSH alone increased (P less than 0.01) FSH and estradiol receptors 3-fold and 4-fold, respectively, over controls by 12 and 24 h. Cyanoketone prevented these increases in FSH and estradiol receptors. Estradiol replacement fully reversed the effects of cyanoketone on FSH action. Replacement with progesterone and testosterone was able to only partially restore levels of FSH receptors; however, estradiol receptor numbers were also increased.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of gonadotropins and granulosa cell secretions on the maturation and fertilization of rat oocytes in vitro

Fully grown germinal vesicle-stage oocytes are induced to resume meiosis and acquire the capacity to undergo fertilization in response to a surge of gonadotropins. The present study examined possible direct and indirect roles of gonadotropins in the maturation and fertilization of rat oocytes by determining 1) the effect of exogenous administration of gonadotropins (priming) to immature rats prior to oocyte collection on the capacity of oocytes to undergo maturation and fertilization in vitro, 2) the effect of follicle-stimulating hormone (FSH) in the maturation media on the resumption of meiosis and subsequent capacity of oocytes to undergo fertilization, and 3) the capacity of oocytes to undergo maturation and fertilization following culture in preovulatory follicular fluid or in conditioned media obtained from gonadotropin-stimulated granulosa cell (GC) cultures. In the first experiment, oocytes from unprimed rats underwent spontaneous meiotic maturation in vitro and 17% underwent subsequent fertilization. Priming increased the proportion of oocytes undergoing fertilization. Maturation of oocytes in media supplemented with various concentrations of FSH or for various lengths of time (6-16 h) in medium with 500 ng FSH/ml indicated that FSH slowed the rate of meiotic maturation, but had no effect on the capacity of the oocytes to be fertilized. Oocytes obtained from primed animals and cultured in the presence of preovulatory follicular fluid were fertilized in proportions similar to those cultured in serum-containing medium. In the third experiment, medium conditioned by FSH-stimulated GC for 40 h slowed the rate of meiotic maturation; the addition of luteinizing hormone (LH) to the FSH-stimulated cells produced a medium in which the rate of oocyte maturation was not different from that of control oocytes (in medium from unstimulated cells). Medium conditioned by FSH- or LH-stimulated GC, but not fibroblasts, increased the proportions of oocytes undergoing fertilization following maturation in those media. FSH + LH stimulation of GC increased the fertilization of oocytes to proportions significantly higher than with either gonadotropin alone. These data suggest that GC respond to gonadotropin stimulation by providing a factor(s) that regulates the rate of oocyte maturation and promotes the capacity of oocytes to undergo fertilization.     

Effects of gonadotropins on oocyte maturation and progesterone production by porcine ovarian follicles cultured in vitro

Effects of gonadotropins on the maturation of isolated oocytes and production of progesterone by porcine ovarian follicles from gonadotropin treated gilts have been studied in vitro. The addition of gonadotropins (2 I. U./ml, PMSG, HGC or 2 mg/ml FSH) to the culture medium resulted in increasing the number (84 - 90 %) of isolated oocytes which reached metaphase II. Expansion of the whole cumulus mass was observed only in media containing PMSG, whereas FSH or HCG alone did not cause these marked changes in the cumulus cells. Denudation of the eggs prior to culture gave no significant differences in the maturation rates between oocytes cultured in media with or without gonadotropins. In vitro maturation of follicle-enclosed oocytes took place only in HCG treated animals. Removing the ovary at 15 or 60 minutes after intravenous HCG administration induced oocyte maturation only in 22% and 17% respectively. A sharp increase in the number of oocytes which resume meiosis during follicle culture was observed 4 hours after HCG injection (84 %) and all of the oocytes of the gilts ovariectomized at 8 hours after HCG injection matured during the culture period. The progesterone production of isolated follicles from control gilts (only PMSG injected) increased slowly during a 96-hour culture period (from 48 to 240 ng progesterone/follicle), whereas the secretion of progesterone was drastically increased after a 15 minute interval between HCG injection and ovariectomy (from 42 to 950 ng progesterone/follicle). Follicles removed 24 hours after HCG injection showed a further increase in steroid production (2000 ng progesterone/follicle) and consistently secreted large amounts of progesterone during the culture period.