Low Doses Of FSH Research Articles

Pre-culture with transferrin-Fe3+ before in vitro maturation improves the developmental competence of porcine oocytes matured in vitro.

Since the developmental competence of oocytes cultured after in vitro maturation (IVM) is low, it is necessary to improve the IVM method for efficient offspring production. In this study, we revealed that transferrin (TF)-Fe3+ was accumulated in follicular fluid with increasing the follicular diameter, and that TF receptor (TFR1) was localized in granulosa cells of pig. Thus, we hypothesized that TF-Fe3+ would be a factor in the induction of developmental competence of porcine oocytes. To mimic the follicular development environment, cumulus-oocyte complexes (COCs) were cultured in pre-IVM medium (low dose of FSH) without or with Holo-TF (monoferric or diferric TF) or Apo-TF (non-iron bond TF). After pre-IVM without or with Holo-TF, COCs were cultured in IVM medium (high dose of FSH and EGF) without or with Holo-TF. Cultivation with Holo-TF increased the expression of follicular development maker (Cyp19a1 and Ccnd2), E2 production, and proliferative activity of cumulus cells, whereas cultivation with Apo-TF did not show these positive effects. The treatment with Holo-TF during pre-IVM, but not during IVM, dramatically induced oocyte maturation with increasing the blastocyst rate. We succeeded in showing for the first time that the cultivation with Holo-TF in pre-IVM can produce embryos in pig with high efficiency.

PSVII-26 Follicular growth in beef cows when using a low dose of FSH and hCG at the time of CIDR removal in a 7-day co-synch + CIDR protocol

Abstract The aim of the study was to compare the growth of the periovulatory follicle in crossbreed cows receiving either a low dose of hCG (150 IU; Chorulon®, Merck) or a low dose of FSH (20 mg)Folltropin®, Vetoquinol) vs. a control group when applied at CIDR removal during an estrus synchronization protocol. At d0, a CIDR (Eazi-Breed CIDR®, Zoetis) was placed, a dose of GnRH (Factrel®, Zoetis) was applied i.m. and transrectal ultrasound was conducted to evaluate cyclicity status. On d7, cows (n = 79) were randomly assigned to three treatments (Control n = 27, FSH n = 26, and hCG n = 26) according to body condition score (1 to 9 scale) and cyclicity status. On the same day, CIDR was removed, a dose of prostaglandin analog (Lutalyse HighCon Injection®, Zoetis) was administered i.m. and an Estrotect™ patch was placed between the hip and tail head. Transrectal ultrasound was conducted to evaluate the diameter of the preovulatory follicle (DPF) at d7 and every 12h from d8 until ovulation was detected. Five cows that did not ovulate were removed from the study. Data were analyzed using GLIMMIX and LIFETEST procedures (SAS). The diameter at ovulation was not different among treatments (Control: 13.02 ± 0.53 mm; FSH: 12.38 ± 0.54 mm; hCG; 11.69 ± 0.47 mm; P = >0.10). Also, the time to ovulation was not different among treatments (P = 0.65; Figure 1a). However, cows of the control group presented estrus signs earlier than FSH and hCG groups (P = 0.02, Figure 1b). In conclusion, the use of a low dose of hCG or FSH at the time of CIDR removal did not increase the DPF or the time to ovulation.

PSXII-36 Use of FSH or hCG to enhance follicular growth and pregnancy per artificial insemination during a split-AI program in crossbreed beef heifers

Abstract The objective of the study was to compare follicle growth and pregnancy per artificial insemination (P/AI) in crossbreed heifers receiving either a low dose of hCG (150 IU) or a low dose of FSH (20 mg) vs. a control group during the final stage of a split-AI protocol. A total of 387 heifers were enrolled in the trial and were randomly assigned in three treatments (Control n = 134, FSH n = 125, and hCG n =128) within reproductive tract score (RTS; 1 to 5 scale), body condition score (BCS; 1 to 9 scale) and pubertal status. All groups were synchronized with a 7-day CO-Synch + CIDR protocol, at CIDR removal the two treatments were injected, an Estrotect patch was placed, and the diameter of the dominant follicle was assessed through transrectal ultrasonography, at day 9 heifers that presented estrus (> 50% of the Estrotect patch rubbed off) were AI, and those that did not were TAI at day 10. An ultrasound was carried out along with the AI to measure the diameter of the preovulatory follicle and 30 days after for pregnancy check. The data was analyzed using the GLIMMIX procedure (SAS). The diameter of the preovulatory follicle was different among treatments (Control: 11.48 ± 0.21; FSH: 11.48 ± 0.25; hCG; 10.73 ± 0.28; P = 0.01). However, the rate of follicular growth (Control: 4.96 ± 0.37; FSH: 4.59 ± 0.39; hCG: 5.12 ± 0.38; P = 0.64) and P/AI (control: 41.35%; FSH: 35.77%; and hCG: 34.38%; P = 0.47) were not different among groups. There was a significant difference (P < .0001) in P/AI when comparing animals that presented estrous (48.99%) or not (29.27%). In conclusion, the use of low dosed of hCG or FSH at the time of device removal did not increase the proportion of pregnant heifers.

The Outcome and Economic Viability of Embryo Production Using IVF and SOV Techniques in the Wagyu Breed of Cattle.

The Japanese black cattle breed (Wagyu) has an improved metabolism, which allows them to have a higher marbling score when compared with other cattle breeds. However, this may affect other aspects of the animal’s physiology, including hormone secretion and their reproductive success, such as their response to synchronization protocols and embryo production. Therefore, the objectives of this study were to test a superovulation protocol (SOV) developed with low doses of FSH and to evaluate the outcome and economic viability of embryo production using the SOV and in vitro fertilization (IVF) approaches in the Wagyu cattle breed. For that, ten Wagyu cows were submitted to five SOVs over a period of 15 months using a standard protocol: CIDR + 3 mg estradiol benzoate (D0), 35 mg FSH (Folltropin®) a.m. and p.m. (D4), 35 mg Folltropin® a.m. and 20 mg p.m. (D5), 20 mg Folltropin® a.m. and 10 mg p.m. (D6), 10 mg Folltropin® and 0.5 mg cloprostenol, both a.m. and p.m., + CIDR removal (D7), 0.05 mg GnRH + insemination 12 and 24 h after (D8) and embryo collection + 0.5 mg of cloprostenol (D16). Thirty days after each SOV, a follicular aspiration was conducted to produce IVF embryos without any pre-synchronization using standard semen in the same group of animals. The average number of embryos produced was 7.63 ± 5.61 (SOV) and 4.52 ± 2.44 (IVF) (p = 0.303). There was no significant correlation between the number of embryos produced by the different techniques (SOV and IVF), indicating that cows that respond well to SOV did not respond well to IVF and vice versa (r = 0.379, p = 0.529). The total cost of each embryo produced by SOV was R$215.00 and R$410.00 for IVF. Therefore, cows that produce less than five embryos by SOV are not economically viable due their lack of response to FSH, and the use of IVF in those animals may be more effective.

Effect of low doses of FSH and P4 on cystogenesis-induced mice as a polycystic ovary syndrome model

Effect of low doses of FSH and P4 on cystogenesis-induced mice as a polycystic ovary syndrome model

Oocytes are required for the preantral granulosa cell to cumulus cell transition in mice

Preantral granulosa cells (PAGCs) differentiate into cumulus cells following antrum formation. Cumulus cells, but not PAGCs, are competent to undergo expansion. Experiments reported here tested the respective roles of both oocytes and FSH in the transition of preantral granulosa cells to cumulus cells competent to undergo expansion. PAGC–oocyte complexes were cultured with or without a low dose of FSH (0.005 IU/ml) and isolated PAGCs were cultured with or without oocytes. At the end of culture, complexes or isolated PAGCs were tested for their ability to undergo cumulus expansion and upregulate expansion transcripts in response to EGF or FSH (0.5 IU/ml). The ability to undergo expansion in response to EGF required the presence of oocytes but not FSH during the culture period. Likewise, complexes isolated from the ovaries of hypogonadal mice, which lack circulating gonadotropins, underwent expansion in response to EGF, but not FSH. In contrast, the ability to activate MAPK3/1 and MAPK14 and undergo expansion in response to FSH required prior exposure to low doses of FSH. However, these low levels (0.005 or 0.025 IU FSH/ml) suppressed expression of Slc38a3 and Amh, two transcripts highly expressed in cumulus cells, suggesting opposing effects of FSH on cumulus cell differentiation. In conclusion, the ability to undergo expansion in response to FSH requires prior exposure to FSH during development, while oocyte-derived factors alone are sufficient to promote the ability to undergo expansion in response to EGF. These results highlight the crucial role of oocytes in driving the differentiation of PAGCs into cumulus cells during the preantral to antral follicle transition.

Adjunctive Use of an Oral Contraceptive (OC) and a Reduced Dose of a GnRH Antagonist for COH/IUI

In preparation for IVF, a long GnRH agonist protocol provides for scheduling of procedures, synchronization of follicles, prevention of premature luteinization (PL), and prevention of premature ovulation of mature follicles. Although COH/IUI cycles are commonly started day 2 of the cycle, selection of the dominant follicle may already be underway. Pretreatment with an OC synchronizes follicles and has been shown to block the occurrence of an LH surge in a subsequent COH cycle (Gonen Y, Fertil Steril 1990). A study with COH/IUI (Manzi DL, Fertil Steril 1995) has shown that about one-third of cycles demonstrate PL, with a very low success rate and a high rate of miscarriage and improved RESULTS with use of a GnRH agonist in the next cycle. We therefore chose to evaluate COH/IUI following OC, with half of the usual dose of the GnRH antagonist, ganirelix (G) to prevent PL and premature release of follicles. Pilot trial. Thirteen women (mean age and body weight 34.5 and 136.5) with unexplained infertility or male factor with weight between 110 and 180 pounds and a day 3 FSH less than 10 IU/ml. having their first COH/IUI cycle took OC for 14 to 21 days. Beginning on the fourth day after OC, rFSH (follitropin beta), 75-225 IU was self-administered daily using a multidose pen. G, 0.125 mg, was administered each morning starting at a lead follicle mean diameter of 14 mm. HCG was given in the evening when there were at least two follicles of 17 mm mean diameter or greater, and IUI was done on the following two mornings. The mean starting dose of rFSH was 161.5 IU. G was given for a mean of 3.5 days. HCG was given after 10.1 days of stimulation with 5.9 follicles over 12 mm. Five women have ongoing pregnancies (38.5%). UTZ showed two sacs in three women, with one of those evolving to one viable fetus. Serum LH levels will be assessed. A high rate of ongoing pregnancy was achieved in this small ongoing study with COH preceded by OC and with adjunctive use of a GnRH antagonist. The high number of follicles and twin implantations are consistent with a synchronizing effect of OC and excellent oocyte quality. Based on this initial experience, we have reduced the starting dose to 75-100 IU. The low dose of FSH should offset the additional cost of G. The use of OC is also helpful in scheduling procedures around career activities. A large prospective randomized trial will be necessary to demonstrate a better clinical outcome compared with COH alone.

Gonadotropin therapy for the treatment of anovulation and for ovarian hyperstimulation for IVF

Knowledge of the mechanisms of single dominant follicle selection has led to the development of a novel and effective ovulation induction regimen for anovulatory women; the step down protocol. This commences with a fixed high gonadotropin dose followed by several decremental steps. For some patients the initial dose is too high, risking ovarian hyperstimulation syndrome. A major improvement to this approach would, therefore, be the ability to use initial screening characteristics to assess the individual FSH threshold beforehand. For IVF treatment, interfering in the process of single dominant follicle selection in ovulatory women by late follicular phase administration of low doses of FSH may result in a significantly reduced duration of stimulation and amounts of exogenous FSH preparations used. Less monitoring would be required and chances for short-term complications or long term risks may be reduced.

Evidence for the role of follicle stimulating hormone in maintaining the oocyte meiotic arrest in pre-antral follicles

The study was designed to clarify the physiological role of FSH in maintaining the meiotic arrest in vitro of oocytes derived from intact and from hypophysectomized hamsters. The sensitivity to FSH in maintaining the meiotic arrest of oocytes during incubation of preantral follicles of adult golden hamsters was estimated: log doseresponse curves were characterized by an ED50 of about 0.2 pg FSH ml (= 1 μ U) . After hypophysectomy oocytes of the pre-antral follicles were screened for the presence of a germinal vesicle (GV). The breakdown of the GV (GVBD) rose from 0 on day 0 to 48.8 ± 13.8% on day 1, to 55.9 ± 6.8% on day 2 and to 64.3 ± 7.4 on day 6 (p < 0.001 vs day 0). Incubation without FSH of the pre-antral follicles obtained from the same hypophysectomized animals showed an almost complete disappearance of the GV at the same time intervals. Incubation with 2 pg FSH ml (= 10 μ U) still demonstrated the inhibiting effect of FSH on the meiotic resumption: 13.4 ± 4.3% (day 0), 36.9 ± 16.3% (day 1), 38.7 ± 21.0% (day 2) and of 38.1 ± 12.6% (p > 0.1) on day 6. These results demonstrate that very low doses of FSH are able to maintain the meiotic arrest in oocytes of pre-antral follicles of the golden hamster and that deprivation of FSH by hypophysectomy involves a disappearance of GV oocytes to a certain extent.

Application of a CHO cell line transfected with the human FSH receptor for the measurement of specific FSH receptor activation inhibitors in human serum

Effects of FSH on ovarian follicular development can be modulated by factors present in serum or by locally produced factors in follicular fluid. Some of these factors may act directly on the FSH receptor. A Chinese hamster ovary cell line (CHO-F3B4) stably transfected with the human FSH receptor has been used to measure the effects of these modulators on FSH-stimulated adenylate cyclase activity. After incubation of CHO-F3B4 cells with human recombinant FSH (recFSH) for 4 h, cAMP levels were elevated 100-230 times above basal levels (ED50 24.9 mU/ml recFSH). cAMP production was inhibited after the addition of increasing amounts (up to 90% of the incubation volume) of hypogonadotrophic human serum (HS) at a fixed stimulatory dose of 30 mU/ml recFSH. At 10% HS the cAMP response was diminished to approximately 40-60% of the original value, whereas at a concentration of 90% HS the cAMP values were diminished to 30%. Effects of serum components on cell viability could be excluded, since forskolin- and cholera-toxin-stimulated cAMP production were not affected by preincubation of the cells in the presence of HS. The FSH-stimulated oestradiol production in rat Sertoli cells, which has been used frequently for in vitro bioassays of FSH, was almost completely inhibited by the addition of human serum, suggesting that serum has more pronounced effects on events downstream of receptor activation. Various specific FSH binding inhibitors have been demonstrated by radioreceptor assays to be present in serum. In order to assess whether such FSH receptor binding inhibitors would also inhibit receptor activation, the specific conditions used in the radioreceptor assays (buffers of low ionic strength) were also used to measure the effects of serum on FSH receptor activation. Under these conditions (a low-salt buffer, corrected for low osmolarity with 200 mM sucrose), CHO-F3B4 cells responded to FSH stimulation in a similar way to that observed in normal buffers. When CHO-F3B4 cells were incubated in this low-salt buffer with a fixed low dose of FSH (3 mU/ml), the addition of 3-90% (v/v) dialysed HS inhibited the FSH-stimulated cAMP accumulation to a similar extent to that in standard conditions. The observed inhibition of adenylate cyclase activation by the low-molecular-mass fraction (< 10 kDa) of HS could be attributed to the presence of salts in this fraction, since the addition of PBS in similar concentrations displayed an equal degree of inhibition. It is concluded that the inhibitory effects of serum on FSH-stimulated cAMP production in CHO-F3B4 cells are small, compared with the inhibition of aromatase induction in rat Sertoli cells. The strong inhibition of aromatase in rat Sertoli cells may result from the effects of serum acting on the FSH receptors as well as on other pathways not related to the FSH receptor. Therefore, measurement of aromatase in Sertoli cells is not suitable for the detection of inhibitors of FSH receptor activation. The CHO-F3B4 cells are useful for the measurement of whether inhibition of FSH receptor activation occurs in serum or follicular fluid from patients with disturbed follicle development.

Differential effects of oxytocin on steroid production by bovine granulosa cells

Oxytocin (OT) and its mRNA are expressed at very low levels in granulosa cells from bovine preovulatory follicles isolated before the LH FSH surge and increase dramatically between the surge and ovulation. We have shown previously that OT stimulates progesterone secretion by granulosa cells obtained before, but not after the gonadotropin surge, suggesting that OT may be involved in the follicular/luteal phase shift in steroidogenesis from estradiol/androgen to progesterone. One objective of this study was to determine if OT affects estradiol as well as progesterone production by utilizing culture conditions that maintain estradiol secretion in vitro. A second objective was to determine if OT regulates steroidogenesis by effects on the levels of mRNA for the steroidogenic enzymes involved in progesterone synthesis, cytochrome P450 side-chain cleavage (P450 scc) and 3β-hydroxysteroid dehydrogenase (3β-HSD), or in estradiol production, cytochrome P450 aromatase (P450 arom). Granulosa cells were isolated from bovine preovulatory follicles and cultured for 3 days with or without OT in medium supplemented with either insulin (1 μg/ml) + 1% fetal calf serum (FCS), which maintains basal estradiol secretion, or low doses of FSH (1 and 2 ng/ml) + 1% FCS, a culture condition that maximizes effects of FSH on estradiol secretion. After the first day of culture, OT stimulated progesterone ( P < 0.01) and inhibited estradiol production ( P < 0.01) in both control and FSH-treated cultures. In contrast, OT had only a small stimulatory effect on the levels of mRNA for P450 scc and 3β-HSD and no effect on mRNA for P450 arom. These findings suggest that: (1) OT plays an autocrine role in regulating the follicular luteal phase shift in steroidogenesis by both increasing progesterone and inhibiting estradiol production and (2) the differential effects of OT on steroid production are not mediated primarily by effects on levels of mRNA for steroidogenic enzymes.

Low and high concentrations of gonadotropins differentially regulate hormone production by theca interna and granulosa cells from bovine preovulatory follicles

Mammalian preovulatory follicles produce primarily estradiol and androgens before the preovulatory gonadotropin surge. In cattle, the LH/FSH surge triggers a rapid decrease in estradiol and androgen production and a dramatic increase in progesterone and oxytocin biosynthesis. It is unclear how changes in gonadotropin concentrations in vivo regulate this follicular/luteal phase shift in hormone production. To address this question, theca interna and granulosa cells were isolated from preovulatory follicles of Holstein heifers approximately 24 h before the expected time of the LH/FSH surge and cultured in defined medium containing insulin (1 microgram/ml), with varying doses of LH or FSH (0, 1, 2, 4, 8, 16, 32, 64, or 128 ng/ml). Media were collected and replaced every 24 h for 3 days, and assayed for androstenedione and progesterone in theca interna cultures, and for estradiol, progesterone and oxytocin in granulosa cell cultures. After the first day of culture (24-72 h), only very low doses of LH (2 or 4 ng/ml) enhanced (p < 0.05) androstenedione secretion by theca interna cultures above control levels, whereas progesterone secretion was increased by a wide range of LH concentrations (p < 0.05), with maximal progesterone secretion at high doses of LH. Likewise, after the first day of culture (24-72 h), estradiol secretion by granulosa cells was stimulated only by low doses of FSH and was inhibited at higher concentrations relative to control cultures (p < 0.05), whereas the production of oxytocin and progesterone was enhanced maximally by high concentrations of FSH (p < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Restoration of Oocyte Maturational Competency during the Nonbreeding Season with Follicle-Stimulating Hormone Stimulation in Squirrel Monkeys (Saimiri Boliviensis Boliviensis)1

The in vitro maturation potential of oocytes retrieved during the nonbreeding season with or without prior in vivo low-dose FSH stimulation was studied in adult squirrel monkeys (Saimiri boliviensis boliviensis). Additionally, the adequacy of various protein supplements in media used for oocyte maturation was investigated. Ovaries were removed from animals in the nonbreeding season (n = 9) with or without prior treatment with a low dose (1 mg) of FSH for four days before ovariectomy. Minimal estradiol elevation was observed in serum even with stimulation. For oocyte collection, ovaries were placed in warmed 21 mM HEPES-buffered Ham's F-10. Oocytes from unstimulated ovaries were retrieved and cultured (47 of 62 recovered) in bicarbonate-buffered Ham's F-10 medium containing 0.5% BSA as protein supplement. Negligible maturation was observed at 48 h (3 of 47; 6%), and no fertilization was seen after insemination. Immature oocytes from animals stimulated with a low dose of FSH were cultured (69 of 94 recovered). With prior FSH stimulation, oocytes placed in 0.5% BSA medium matured (13 of 24; 54%) and fertilized (7 of 21; 33%) in marked contrast to oocytes from the nonstimulated monkeys. Additionally, 20% monkey serum and 20% human follicular fluid were studied as alternative protein supplements for the FSH-pretreated oocytes; these produced similar maturation rates (10 of 22, 45%; 10 of 23, 43%, respectively) and fertilization rates (8 of 21, 38%; 6 of 21, 29%, respectively). In some cases, 2 pronuclei were observed at 16 h and 4 cells were observed at 40 h. Of those activated, 63% showed cleavage ranging from 2 to 8 cells by 96 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Low doses of FSH stimulate follicular growth and maturation in anestrous heifers

Low doses of FSH stimulate follicular growth and maturation in anestrous heifers

Inhibin production by primary Sertoli cell-enriched cultures: regulation by follicle-stimulating hormone, androgens, and epidermal growth factor.

Inhibin levels in the serum-free media of primary Sertoli cell-enriched (SCE) cultures were studied as a function of time and hormonal treatment. SCE cultures were established from 20-day-old rats and maintained in SF media supplemented with insulin, transferrin, epidermal growth factor (EGF), and bacitracin. Radioimmunoassayable inhibin was measured using an antibody directed against a synthetic porcine inhibin-alpha (pI alpha) and measured using this same synthetic peptide as well as a highly purified ovine inhibin standard. Results of these determinations are expressed in terms of a synthetic peptide as femtomoles of pI alpha-(1-26)-G-Y-per ml/10(6) cells. Inhibin levels (+/- SEM) that accumulated at this rate in media from control cultures were 184.9 +/- 6.1 and 167.4 +/- 5.2 on days 0-4 and 4-8, respectively. When FSH (oFSH 17; 1-1000 ng/ml) was added, a dose-dependent increase in inhibin levels was significant at all time points beyond the initial 24 h. The simultaneous addition of 2 x 10(-7) M testosterone (T) with low doses of FSH addition of 2 x 10(-7) m testosterone (T) with low doses of FSH suppressed the inhibin response to FSH, but when T was combined with 300 ng/ml FSH, there was no effect of T on inhibin levels compared to FSH alone, regardless of time in culture. In spite of the modest effect of T, androstenedione (A; 2 x 10(-13) to x 10(-5) M) produced a dose-dependent suppression of inhibin levels. This inhibition was also observed at all doses of FSH. The action of A was not due to its conversion to estrogens, as 17 beta-estradiol had no effect on inhibin production by SCE cultures in either the presence or absence of FSH. The effect of EGF, a component of the basal serum-free medium, was next examined; it produced a 1.5-fold higher level of inhibin (188.7 +/- 9.5) compared to cultures without EGF (135.6 +/- 5.0). When SCE cultures were plated with FSH plus EGF, the stimulation of inhibin levels was additive. We conclude that in Sertoli cell cultures established from immature rats (1) the accumulation of inhibin in medium declines from 90 fmol/10(6) cells.day (initial 24 h) to 40-50 fmol/10(6) cells.day (over the first 48 h) and continues to accumulate in the medium for 8 days of culture; (2) FSH regulates the production of inhibin by Sertoli cells; the best dose response is observed over a 3-day period.(ABSTRACT TRUNCATED AT 400 WORDS)

Growth hormone enhances follicle-stimulating hormone-induced differentiation of cultured rat granulosa cells.

Suppression of serum GH levels in immature rats is associated with delayed onset of puberty and decreased ovarian steroidogenic responsiveness to FSH. To investigate possible direct effects of GH on the differentiation of ovarian cells, granulosa cells from hypophysectomized estrogen-treated rats were cultured with FSH in the presence or absence of GH for 3 days. FSH stimulated granulosa cell LH receptor formation and steroid production in a dose-dependent manner. Concomitant treatment with GH increased LH receptor content by enhancing the action of low doses of FSH without substantial increases in the maximal response. This increase was due to an elevation in the receptor number rather than changes in their affinity for hCG. At 3 ng/ml FSH, concomitant treatment with ovine or bovine GH increased LH/hCG binding in a dose-dependent manner, with 300 ng/ml GH increasing the FSH action by about 3-fold. LH receptors in the GH-treated cells were functional, as indicated by the enhanced cAMP production of these cells in response to LH treatment. The cellular protein content in the FSH-treated cultures was slightly increased by GH (18%), but cell number and viability were unaffected. The change in cell protein content could not account for the increases in the amount of LH receptors. In addition to its effects on LH/hCG receptor content, GH also augmented FSH-stimulated progesterone and 20 alpha-hydroxy-4-pregnen-3-one production in a dose-dependent manner, with 100 ng/ml GH causing significant increases in FSH-induced progesterone production. In contrast, GH treatment did not significantly affect FSH-stimulated estrogen production. The augmentating effects of GH on LH receptor formation and progestin biosynthesis were associated with an enhancement of FSH-stimulated cAMP production. In addition, GH increased forskolin- and 8-bromo-cAMP-induced LH receptor formation and progestin production. Thus, GH-augmented LH receptor induction and progestin biosynthesis may be due to both increased cAMP production and enhanced action of cAMP. The present data have demonstrated that GH augments gonadotropin-stimulated differentiation of ovarian granulosa cells, suggesting an important regulatory role of GH in follicular growth and pubertal development.

Actions of mammalian pituitary gonadotropins (FSH and LH) in reptiles: I. Male snakes

Ovine FSH and LH were tested in hypophysectomized male snakes, Thamnophis sauritus and T. sirtalis. FSH was considerably more potent than LH and appeared capable of stimulating testis growth, spermatogenesis, and interstitial cell activity (androgenesis). The effects of relatively large amounts of LH were comparable to a low dose of FSH in promoting slight spermatogenic activity; LH did not differentially affect steroiodogenesis. Testosterone did not affect spermatogenesis in T. sirtalis. These results confirm previous findings with purified mammalian hormones in lizards, and suggest that all aspects of testicular function in squamate reptiles may be accounted for by a single FSH-like gonadotropin.