Doses Of LH Research Articles

Split daily recombinant human LH dose in hypogonadotrophic hypogonadism: a nonrandomized controlled pilot study

This prospective controlled nonrandomized pilot study was conducted to investigate whether split daily doses of recombinant human LH (rHLH) is more efficacious than the single daily dose in supporting follicular development and ovulation in primary hypogonadotrophic hypogonadism (HH). Twenty-seven women with HH received a 150 IU fixed daily subcutaneous dose of recombinant human FSH, supplemented by 75 IU daily dose of rHLH given either as a single dose (n=9; single-dose group) or four equally divided doses (n=18; split-dose group). Ovulation was defined by three efficacy end points: at least one follicle ⩾17mm in diameter, pre-ovulatory serum oestradiol ⩾400pmol/l and a midluteal progesterone ⩾25nmol/l. Although lacking statistical significance, the proportion of women in the rHLH split-dose group who fulfilled all three end points was higher than the single-dose group (72.2% versus 55.6%). Women in the split-dose group achieved higher serum oestradiol concentrations per follicle, endometrial thickness measurements and numbers of follicles than in the single-dose group (not statistically significant). The odds ratio for ovulation rate was 2.08 (not statistically significant). There were no serious untoward side effects. Administering rHLH in split daily doses could provide superior results compared with the traditional single daily dose.We conducted this clinical study to investigate whether a split daily dose protocol of recombinant human LH (rHLH) is more efficacious than the single daily dose in supporting follicular development and ovulation in primary hypogonadotrophic hypogonadism (HH). HH is an uncommon entity that can lead to very low or undetectable serum gonadotrophin concentrations. It manifests in anovulation, amenorrhoea and subsequent infertility. Twenty-seven women with HH received a 150 IU fixed daily subcutaneous dose of recombinant human FSH, supplemented by a 75 IU daily dose of rHLH given either as a single dose (n=9; single-dose group) or four equally divided doses (n=18; split-dose group). Ovulation was defined by these three efficacy end points: at least one follicle ⩾17mm in mean diameter, pre-ovulatory serum oestradiol concentration ⩾400pmol/l and a midluteal progesterone concentration ⩾25nmol/l. The proportion of women in the rHLH split-dose group who fulfilled all three end points was higher than the single-dose group (72.2% versus 55.6%). Women in the split-dose group achieved higher serum oestradiol concentrations per follicle, endometrial thickness measurements and numbers of follicles than in the single-dose group, without statistical significance. Women who received the split-dose regimen were more likely to have ovulation than the other group. We had no serious problematic side effects. Our results suggest that administering rHLH in split daily doses could provide superior results compared to the traditional single daily dose.

Gene expression differences induced by equimolar low doses of LH or hCG in combination with FSH in cultured mouse antral follicles

In a natural cycle, follicle growth is coordinated by FSH and LH. Follicle growth stimulation in Assisted Reproductive Technologies (ART) requires antral follicles to be exposed to both FSH and LH bioactivity, especially after GNRH analog pretreatment. The main aim was to detect possible differences in gene expression in granulosa cells after exposing the follicle during antral growth to LH or hCG, as LH and hCG are different molecules acting on the same receptor. Effects of five gonadotropin treatments were investigated for 16 genes using a mouse follicle culture model. Early (day 6) antral follicles were exposed to high recombinant FSH combined or not with equimolar concentrations of recombinant LH (rLH) or recombinant hCG (rhCG) and to highly purified human menopausal gonadotropin (HP-hMG) for 6 h, 12 h, or 3 days. Expression differences were tested for genes involved in steroidogenesis: Mvk, Lss, Cyp11a1, Hsd3b1, Cyp19a1, Nr4a1, and Timp1; final granulosa differentiation: Lhcgr, Oxtr, Pgr, Egfr, Hif1a, and Vegfa; and cytokines: Cxcl12, Cxcr4, and Sdc4. Lhcgr was present and upregulated by gonadotropins. Nr4a1, Cxcl12, and Cxcr4 showed a different expression pattern if LH bioactivity was added to high FSH in the first hours after exposure. However, no signs of premature luteinization were present even after a 3-day treatment as shown by Cyp19a1, Oxtr, Pgr, and Egfr and by estrogen and progesterone measurements. The downstream signaling by rhCG or rLH through the LHCGR was not different for this gene selection. Granulosa cells from follicles exposed to HP-hMG showed an enhanced expression level for several genes compared with recombinant gonadotropin exposure, possibly pointing to enhanced cellular activity.

Outcome of in vitro fertilization in patients with proven poor ovarian responsiveness after early vs. mid-follicular LH exposure: a prospective, randomized, controlled study

To compare early vs. mid-follicular exposure to LH in patients with poor ovarian responsiveness undergoing in vitro fertilization (IVF). Prospective, randomized, controlled trial. University Hospital, University-affiliated private Clinic. Five hundred-thirty women with poor ovarian responsiveness during the first IVF cycle, undergoing their second IVF attempt. In a GnRH-analogue long protocol, ovarian stimulation with recombinant FSH (300IU/day) plus randomly assigned addition of recombinant LH (150IU/day) from day 1 (early LH exposure; n = 264) or from day 7 (late LH exposure; n = 266). Primary outcome was the number of oocytes retrieved. Secondary outcomes were: cancellation rate, total gonadotropin dose, duration of ovarian stimulation, number of embryos available for transfer, pregnancy rate per started cycle, per OPU and per embryo transfer, implantation rate, delivered/ongoing pregnancy rate. Apart from the totally administered LH dose, that was significantly higher in the group receiving it from day 1, all parameters related to IVF outcome were non significantly different in the two groups. Adding LH to FSH from day 1 or from day 7 of ovarian stimulation in a GnRH-agonist long protocol exerts comparable effects on IVF outcome in poor responders.

Supplementation of equine early spring transitional follicles with luteinizing hormone stimulates follicle growth but does not restore steroidogenic activity

This study was conducted to test the hypothesis that supplementation of growing follicles with LH during the early spring transitional period would promote the development of steroidogenically active, dominant follicles with the ability to respond to an ovulatory dose of hCG. Mares during early transition were randomly assigned to receive a subovulatory dose of equine LH (in the form of a purified equine pituitary fraction) or saline (transitional control; n = 7 mares per group) following ablation of all follicles >15 mm. Treatments were administered intravenously every 12 h from the day the largest follicle of the post-ablation wave reached 20 mm until a follicle reached >32 mm, when an ovulatory dose of hCG (3000 IU) was given. Saline-treated mares during June and July were used as ovulatory controls. In a preliminary study, injection of this pituitary fraction (eLH) to anestrus mares was followed by an increase in circulating levels of LH (P < 0.01) but not FSH (P > 0.6). Administration of eLH during early transition stimulated the growth of the dominant follicle (Group x Day, P < 0.00001), which attained diameters similar to the dominant follicle in ovulatory controls (P > 0.1). In contrast, eLH had no effect on the diameter of the largest subordinate follicle or the number of follicles >10 mm during treatment (P > 0.3). The numbers of mares that ovulated in response to hCG in transitional control, transitional eLH and ovulatory control groups (2 of 2, 3 of 5 and 7 of 7, respectively) were not significantly different (P > 0.1). However, after hCG-induced ovulation, all transitional mares returned to an anovulatory state. Circulating estradiol levels increased during the experimental period in ovulatory controls but not in transitional eLH or transitional control groups (Group x Day, P = 0.013). In addition, although progesterone levels increased after ovulation in transitional control and transitional eLH groups, levels in these two groups were lower than in the ovulatory control group after ovulation (Group, P = 0.045). In conclusion, although LH supplementation of early transitional waves beginning after the largest follicle reached 20 mm promoted growth of ovulatory-size follicles, these follicles were developmentally deficient as indicated by their reduced steroidogenic activity.

Outcome of gonadotropin therapy for male infertility due to hypogonadotrophic hypogonadism

Data on the management of male infertility secondary to hypogonadotrophic hypogonadism (HH) are limited. We report our extensive experience with intramuscular injections of gonadotropins, one of the two methods used for this purpose. Eighty-seven married men (median age, 28 years) with either congenital (47 men) or acquired (40 men) HH were treated for a median of 26 months (range, 6-57) with intramuscular injections of gonadotropins (HCG/HMG) three times weekly for the purpose of achieving fertility. The outcome was assessed by achievement of one or more pregnancies. Of the 151 courses of HCG/HMG treatment administered to 87 patients, 85 courses (56.3%) were successful, resulting in 85 pregnancies (median pregnancy rate 2, range 1-10) in 35 patients (40%) while 52 cases did not achieve pregnancy. Responders had larger pretherapy testicular volume (9 +/- 3.6 cc) compared to non-responders [(5.7 +/- 2.0 cc), P < 0.0001], but there was no difference in age, LH, FSH or testosterone levels or doses of HCG/HMG used. The pregnancy rate was similar in those with congenital (51.4%) and acquired causes (48.6%) of HH (P = 0.83). Only testicular size was predictive of conception (P < 0.001, odds ratio 1.5, 95% CI 1.21-1.92) while age, pretherapy levels of testosterone, LH, FSH and doses of HCG/HMG did not predict the success of pregnancy. Gonadotropins are moderately effective in achieving one to several pregnancies in HH. Only testicular size is predictive of success in achieving pregnancy. There is no difference in success between those with congenital and acquired causes of HH.

Administration of follitropin alfa and lutropin alfa combined in a single injection: a feasibility assessment

BackgroundGonadotrophins are routinely administered in assisted reproductive technology (ART) treatment protocols. Recombinant human follicle-stimulating hormone (r-hFSH; follitropin alfa) and recombinant human luteinizing hormone (r-hLH; lutropin alfa) can be administered individually or in a fixed combination. The ability to vary the FSH to LH dose ratio in a single injection without compromising the bioactivity of either gonadotrophin or generating losses of active principle is important for physicians and patients alike.MethodsThis study investigated whether follitropin alfa (GONAL-f (R)), as lyophilized powder for reconstitution or solution from the GONAL-f (R) (filled-by-mass [FbM]) Prefilled Pen, could be used to reconstitute Pergoveris TM (follitropin alfa/lutropin alfa 150 IU/75 IU) lyophilized powder. In Ratio Groups 1 and 2, the r-hFSH:r-hLH ratio was 3:1; in Ratio Groups 3 and 4, the ratios of r-hFSH:r-hLH were 5:1 and 8:1, respectively. The protein content and bioactivity of each mixed solution were evaluated. The r-hFSH and r-hLH content was determined using reverse-phase high performance liquid chromatography. The biological activity of r-hFSH and r-hLH was assessed using the Steelman-Pohley and Van Hell in vivo bioassays in rats, respectively.ResultsFollitropin alfa/lutropin alfa 150 IU/75 IU lyophilized powder could be successfully mixed with follitropin alfa 75 IU FbM solution that was either reconstituted from lyophilized powder or injected directly from the prefilled pen to create solutions with ratios of r-hFSH and r-hLH from 3:1 to 8:1. The measured content of r-hFSH and r-hLH corresponded favourably with the target protein content in Ratio Groups 1–4. The in vivo target and measured bioactivity of r-hFSH and r-hLH were also closely matched in all Ratio Groups.ConclusionFollitropin alfa lyophilized powder or solution can be accurately mixed with follitropin alfa/lutropin alfa 150 IU/75 IU lyophilized powder to enable physicians to administer ratios of r-hFSH and r-hLH in the IU range from 3:1 to 8:1. Mixing of recombinant formulations offers flexibility for patients receiving follitropin alfa and lutropin alfa for ART protocols in clinical practice.

Effects of FSH and LH on Steroid Production by Buffalo (Bubalus bubalis) Granulosa Cells CulturedIn VitroUnder Serum-Free Conditions

The objective of this study was to examine the effects of FSH and LH on oestradiol-17β and progesterone production by buffalo granulosa cells cultured under serum-free conditions. Granulosa cells (3 × 10(5) ) from small (≤ 5 mm diameter) follicles were cultured for up to 4 days in 48-well plates coated with 3.3 μg/cm(2) fibronectin in Dulbecco's modified Eagle's medium (DMEM) : nutrient mixture F-12 Ham (1 : 1 ratio) supplemented with 10(-7) m androstenedione, 5 μg/ml human apo-transferrin and 0.1% bovine serum albumin, in the presence or absence of FSH or LH (0, 1, 2, 4, 8, 16, 32 or 64 ng/ml each). Basal oestradiol-17β production by granulosa cells from small follicles reduced (p < 0.01) from days 1 to 2 of culture and became undetectable by day 3 and basal progesterone production increased (p < 0.05) from day 1 through day 4 of the culture. Although there was no effect of FSH on day 1 of the culture, FSH at 2, 4, 8 and 16 ng/ml increased (p < 0.05) oestradiol-17β production by granulosa cells from small follicles on day 2. Progesterone secretion was increased (p < 0.05) by all doses of FSH on all days of culture. All doses of LH had no effect on oestradiol-17β or progesterone production by granulosa cells from small follicles on any day of the culture. The results of this study demonstrate a serum-free culture system for buffalo granulosa cells and stimulatory effect of FSH but not LH on steroid hormone production by buffalo granulosa cells under these conditions.

Estrogen Receptor β Is Required for Optimal cAMP Production in Mouse Granulosa Cells

Granulosa cells of preovulatory follicles differentiate in response to FSH, and this differentiation is augmented by estradiol. We have previously shown that FSH-mediated granulosa cell differentiation requires functional estrogen receptor-beta (ERbeta) by demonstrating that the granulosa cells of ERbeta(-/-) FSH-treated mice are unable to maximally induce expression of the LH receptor (an indicator of granulosa cell differentiation) compared with ERbeta(+/+) controls. As a result, FSH-primed ERbeta(-/-) granulosa cells exhibit a reduced response to a subsequent ovulatory dose of LH. In this study, we further characterized the attenuated response of ERbeta(-/-) granulosa cells to stimulation by LH and FSH using isolated mouse granulosa cells and primary granulosa cell cultures. We observed a 50% reduction in cAMP levels in cultured ERbeta(-/-) granulosa cells exposed to LH compared with ERbeta(+/+) controls. We also observed an attenuated genomic response in granulosa cells isolated from FSH-primed ERbeta(-/-) mice compared with ERbeta(+/+) controls. Our data indicate that this attenuated response may result from inadequate levels of cAMP, because cAMP levels in cultured ERbeta(-/-) granulosa cells exposed to forskolin were approximately 50% lower than in ERbeta(+/+) granulosa cells. Phosphorylation of cAMP regulatory element binding protein, an indicator of protein kinase A activity, was also reduced in FSH-treated ERbeta(-/-) granulosa cells compared with ERbeta(+/+) controls. These are the first data to indicate that ERbeta plays a role in the induction of the cAMP pathway in mouse granulosa cells and that disruption of proper ERbeta signaling associated with this pathway may cause negative effects on ovulation and fertility.

175 FOLLICULAR DIAMETER AND OVULATION RATE IN NELLORE COWS TREATED WITH LH

The aim of the present experiment was to verify the correlation between follicular diameter and ovulation rate in Nellore cows submitted to LH treatment to add information to the limited literature about this subject. A preliminary test (experiment 1) was performed to determine the lowest dose of LH necessary to induce ovulation in follicles with diameter ≥10 mm. At a random stage of the estrous cycle (D0), Nellore cows (n = 20) received a progesterone intravaginal device (1.0 g, Primer®, Tecnopec, Sao Paulo, Brazil) and 2.5 mg of estradiol benzoate (EB, i.m., Estrogin®; Farmavet, Sao Paulo, Brazil). Eight days later (D8) PGF2α was administered (150 μg of D-cloprostenol, i.m., Prolise® ARSA S.R.L., Buenos Aires, Argentina) and the device was removed. Twenty-four hours after device removal, cows were treated with EB (1.0 mg, i.m.) and 48 h afterward; ovulation was determined by ultrasonography (US, Aloka 900, Tokyo, Japan). Three days after ovulation, the follicular growth was daily observed by US, and when the follicles reached ≥10 mm diameter, the cows were randomly allocated in 4 groups and treated with different doses of LH (i.m., Lutropin-V®, Bioniche, Ontario, Canada): Groups 1.56; 3.12; 6.25, and 12.5 mg. The data were analyzed by Fisher’s Exact Test (PROC FREQ, SAS). The ovulation rates were 0% (0/5), 80% (4/5), 80% (4/5), and 80% (4/5) respectively, and it was lower in group 1.56 when compared with the others (P &lt; 0.02). Taking in account the results above, the dose of 6.25 mg to induce ovulation in Nellore cows (n = 58, experiment 2) submitted to the same protocol used in experiment 1 and randomly allocated in 5 groups according to follicular diameter at the time of LH administration: G1 (7.0–7.5 mm), G2 (7.6–8.0 mm), G3 (8.1–8.5 mm), G4 (8.6–9.0 mm), and G5 (9.1–10.0 mm). The results were analyzed by Logistic Regression (PROC GENMOD, SAS). The ovulation rates were 0% (0/11), 20% (2/10), 36% (4/11), 36% (4/11), and 90% (9/10), respectively, for G1, G2, G3, G4, and G5. When the data from 2 groups (except G5) were combined, G1 + G2 (7.0–8.0 mm), G3 + G4 (8.1–9.0 mm), and G5 (9.1–10.0) ovulation rates were, respectively, 9% (2/21), 36% (8/22), and 90% (9/10). There were significant differences when comparing G1 + G2 v. G5 (P &lt; 0.01), G3 + G4 v. G5 (P &lt; 0.02) and G1 + G2 v. G3 + G4 (P &lt; 0.03). After LH application, the smallest follicle that ovulated had a diameter of 7.65 mm. In summary, although LH administration induced ovulation in one follicle of 7.65 mm, high ovulation rates were observed only when the follicles reached &lt;9.0 mm. These data will be used to study the correlation between ovulation rate and expression of isoforms of LH mRNA receptors in theca and granulosa cells. Fellowship from *FAPESP (Sao Paulo, Brazil) and **CAPES (Brasilia, Brazil).

Superovulation and in vitro oocyte maturation in three species of mice ( Mus musculus, Mus spretus and Mus spicilegus)

Mouse oocytes can be obtained via superovulation or using in vitro maturation although several factors, including genetic background, may affect response. Our previous studies have identified various mouse species as models to understand the role of sexual selection on the evolution of sperm traits and function. In order to do comparative studies of sperm–oocyte interaction, we sought reliable methods for oocyte superovulation and in vitro maturation in mature females of three mouse species (genus Mus). When 5 IU pregnant mare's serum gonadotrophin (PMSG) and 5 IU human chorionic gonadotrophin (hCG) were injected 48 h apart, and oocytes collected 14 h post-hCG, good responses were obtained in Mus musculus (18 ± 1.3 oocytes/female; mean ± S.E.M.) and Mus spretus (12 ± 0.8), but no ovulation was seen in Mus spicilegus. Changes in PMSG or hCG doses, or longer post-hCG intervals, did not improve results. Use of PMSG/luteinizing hormone (LH) resulted in good responses in M. musculus (19 ± 1.2) and M. spretus (12 ± 1.1) but not in M. spicilegus (5 ± 0.9) with ovulation not increasing with higher LH doses. Follicular puncture 48 h after PMSG followed by in vitro maturation led to a high oocyte yield in the three species ( M. musculus, 23 ± 0.9; M. spretus, 17 ± 1.1; M. spicilegus, 10 ± 0.9) with a consistently high maturation rates. In vitro fertilization of both superovulated and in vitro matured oocytes resulted in a high proportion of fertilization (range: 83–87%) in the three species. Thus, in vitro maturation led to high yields in all three species. These results will allow future studies on gamete interaction in these closely related species and the role of sexual selection in gamete compatibility.

Influence of different concentrations of LH and FSH on in vitro caprine primordial ovarian follicle development

The roles of gonadotrophins in the regulation of primordial ovarian follicular development remains unclear. The aims of the present study were to investigate the effects of LH alone or in combination with FSH on the survival, activation and growth of in vitro caprine primordial follicles, using histology and transmission electron microscopy (TEM). To this end, samples of the caprine ovarian cortex were cultured for 1 or 7 days (39 °C), in an atmosphere of 5% CO 2, in minimum essential medium (MEM—control medium) supplemented with different concentrations of LH (0, 1, 5, 10, 50 or 100 ng/ml—Experiment 1). In Experiment 2, the control medium was supplemented with FSH (50 ng/ml) and the different concentrations of LH (0, 1, 5, 10 or 50 ng/ml also used). Small samples of non-cultured ovarian tissue, as well as those cultured for 1 or 7 days in a specific medium were processed for classical histological evaluation and TEM to evaluate the follicular integrity and to calculate the percentage of normal follicles. Additionally, the effects of FSH on the oocyte and follicle diameters of the cultured follicles were evaluated. Results showed that after 7 days of culture, the presence of LH (10, 50 or 100 ng/ml) in the culture media significantly increases the percentage of developing follicles ( P < 0.05). In addition, fragments cultured in media supplemented with FSH alone or FSH plus LH had a higher percentage of developing follicles after 1 and 7 days of culture, compared to the control ( P < 0.05). At the end of culture period, 1 and 5 ng/ml LH increased the follicular diameter, while the addition of 1, 5 or 10 ng/ml LH increased the oocyte diameter ( P < 0.05). A combination of FSH with 1 and 5 ng/ml LH increased the follicular diameter. With regard to follicle survival, low doses of LH (1 ng/ml) maintained follicle viability—similar to day 0 and in the control medium after 7 days of culture. However, higher concentrations of LH (5, 10, 50 and 100 ng/ml) induced atresia in the goat follicles. Culture of the ovarian cortex for 7 days in medium supplemented with FSH alone or FSH plus 1 ng/ml LH kept the ultrastructural characteristics of follicles similar to that of day 0. In conclusion, it can be said that, the addition of low concentrations of LH (1 ng/ml) combined with or without FSH maintained the goat follicular ultrastructural integrity, but LH in doses higher than 5 ng/ml induced atresia in in vitro goat preantral follicles.

Evaluation of endocrine testing of Leydig cell function using extractive and recombinant human chorionic gonadotropin and different doses of recombinant human LH in normal men

The functional testing of endocrine testis uses extractive human chorionic gonadotropin (ehCG). Recombinant human hCG (rhCG), avoiding any contamination, should replace ehCG. Moreover, a functional evaluation with recombinant human LH (rhLH) would be closer to physiology than a pharmacological testing with hCG. The study was conducted in normal men. We first evaluated the dose-effect of ehCG on plasma testosterone and estradiol levels, before and after injection of either hCG or vehicle. Secondly, the responses to the optimal dose of ehCG were compared with those of rhCG. Thirdly, we investigated the dose-effect of rhLH, on steroid hormone secretion. LH, testosterone, and estradiol plasma levels were measured after the injection of either rhLH or placebo. ehCG induced dose-dependent increases in plasma estradiol and testosterone levels. They respectively peaked at 24 and 72 h after the injection. The most potent dose of ehCG (5000 IU) induced results similar to those observed with 250 microg (6500 IU) rhCG. By comparison with placebo, rhLH induced a significant and dose-dependent increase in plasma testosterone levels 4 h after the injection. Peak response of testosterone to rhLH and rhCG was significantly correlated. rhLH did not induce significant change in plasma estradiol level. In normal men, a single i.v. injection of 150 IU rhLH induces a 25% rise in plasma testosterone levels by comparison with placebo. At the moment, the dynamic evaluation using hCG remains the gold standard test to explore the Leydig cell function. The use of 250 microg rhCG avoiding any contamination should be recommended.

Effects of an Early Postnatal Treatment of Hypogonadotropic Hypogonadism with a Continuous Subcutaneous Infusion of Recombinant Follicle-Stimulating Hormone and Luteinizing Hormone

The neonatal-midinfancy surge in pulsatile gonadotropin secretion is attributable to an increase in GnRH pulse amplitude and is associated with a rapid expansion of Leydig and Sertoli cell populations with concomitant surges in testosterone, inhibin, and anti-Mullerian hormone production as well as an increase in testicular volume. Boys with congenital hypogonadotropic hypogonadism (HH) do not activate these processes. A potential cause for azoospermia and infertility in adult life is deficient proliferation of immature Sertoli cells before and during puberty due to the absence of FSH. The objective of the study was to investigate whether early postnatal continuous sc infusion of gonadotropins could mimic the physiological growth of testes and to evaluate responses of the Leydig and Sertoli cells to early gonadotropin replacement. Two neonates (P1 with hypotuitarism and P2 with HH) with micropenis and microorchidism were treated for 6 months with high doses of recombinant LH and FSH (a gift of Luveris and Gonal-F from Serono, Lyon, France) delivered sc with an insulin pump. Gonadotropin continuous sc infusion increased mean serum LH and FSH to normal or supranormal levels. Mean testosterone increased from undetectable levels to 7.6 and 5.2 nmol/liter, respectively, in P1 and P2. Inhibin B and anti-Müllerian hormone increased to normal levels. Mean testicular volume increased from 0.45 to 0.57 ml at birth to 2.10 ml at 7 months. Stretched penile length increased from 8 to 30 mm (P1) and 12 to 48 mm (P2). The present regimen induced physiological postnatal testes growth and high-normal activation of Leydig and Sertoli cells.

Oral contraceptive pretreatment in women undergoing controlled ovarian stimulation in ganirelix acetate cycles may, for a subset of patients, be associated with low serum luteinizing hormone levels, reduced ovarian response to gonadotropins, and early pregnancy loss

Oral contraceptive pretreatment facilitated scheduling of pure FSH/GnRH antagonist cycles but in a small subset of patients was associated with low serum LH levels, reduced ovarian response, and early pregnancy loss. Supplementation with LH could be examined as a possible way to improve cycle outcome.

Transport of Preimplantation Embryos in the Genitalia of Buffalo Heifers Superovulated with pFSH and Variable Doses of LH

Transport of Preimplantation Embryos in the Genitalia of Buffalo Heifers Superovulated with pFSH and Variable Doses of LH

Development of a cryopreservation protocol for Leydig cells

In the present study, we describe a procedure to cryopreserve the postnatal members of the Leydig cell lineage, including progenitor (PLC), immature (ILC) and adult (ALC) Leydig cells from, respectively 21-, 35- and 90-day-old rats. The cells were resuspended in a culture medium supplemented with 1% bovine serum albumin (Dulbecco's Modified Eagle's Medium [DMEM]/F12) to a final concentration of 2 x 10(6)cells/ml and the effects of varying concentrations of dimethylsulfoxide (DMSO) (5, 10, 15 or 20%) were assessed after freezing at -70 degrees C and then storing in liquid nitrogen. After 12 months of frozen storage, these cells were thawed rapidly at 37 degrees C and Trypan Blue exclusion staining and attachment to culture dishes were assessed as measures of viability. The trypan blue exclusion and attachment rates for Leydig cell stages were around 85% in the presence of 15% DMSO. After frozen storage, Leydig cell steroidogenic capacity in response to a range of LH doses, (0.01-100 ng/ml) was unchanged compared with freshly isolated control cells. Furthermore, the steady-state mRNA levels for Leydig cell specific transcripts were maintained. This study demonstrates that purified rat Leydig cells at a range of developmental stages can be frozen and that the cryopreserved cells retain normal function.

The Effect of the Presence and Pattern of Luteinizing Hormone Stimulation on Ovulatory Follicle Development in Sheep1

The aim of this study was to examine the role of LH on the growth of the large preovulatory follicle and its secretion of hormones in sheep. Ewes with ovarian autotransplants were treated with GnRH-antagonist at the time of luteal regression and different LH regimes applied for 60-66 h before administration of an ovulatory stimulus (hCG). In Experiment 1 (N = 24; n = 8), ewes received either no LH or constant or pulsatile infusion of LH at the same dose (1.25 microg/h). In Experiment 2 (N = 12, n = 6), LH was constantly infused at a rate of 1.25 microg or 2.5 microg oLH/h. In Experiment 1, animals receiving either pulsatile or constant LH exhibited increases in estradiol and inhibin A secretion (P < 0.001) and a depression in FSH (P < 0.001) that resembled the normal follicular phase. Similarly in Experiment 2, doubling the dose of LH resulted in a two-fold increase in ovarian estradiol secretion (P < 0.05) but no other changes. All animals receiving LH, regardless of the pattern of stimulation, ovulated and established a normal luteal phase. In contrast, no LH treatment resulted in constant immuno-active LH without pulses, unchanged FSH and inhibin A concentrations (P < 0.05), and basal estradiol secretion (P < 0.001). Morphologically normal large antral follicles were observed in this group and although corpora lutea formed in response to hCG, progesterone profiles were abnormal. In conclusion, these results suggest that LH is an essential requirement for normal ovulatory follicle development and subsequent luteal function and show that a pulsatile mode of LH stimulation is not required by ovulatory follicles.

Immunization of pigs against chicken gonadotropin-releasing hormone-II and lamprey gonadotropin-releasing hormone-III: Effects on gonadotropin secretion and testicular function1

The objective of this experiment was to evaluate the effects of active immunization against 2 GnRH isoforms on gonadotropin secretion and testicular function in pigs. Synthetic chicken (c) GnRH-II and lamprey (l) GnRH-III peptides, with the common pGlu-His-Trp-Ser sequence at the N-terminal omitted, were conjugated to BSA. Forty-eight male piglets were randomly assigned to 1 of 4 treatments. Pigs on treatment 1 were actively immunized against cGnRH-II, whereas pigs on treatment 2 were actively immunized against lGnRH-III. Control pigs on treatment 3 were actively immunized against the carrier protein (BSA), and pigs on treatment 4 were castrated and actively immunized against BSA. The BSA conjugate was emulsified in Freund's Incomplete Adjuvant and diethylaminoethyldextran. Primary immunization was given at 13 wk of age (WOA) with booster immunizations given at 16 and 19 WOA. Body weight and plasma samples were collected weekly beginning at 11 WOA. Treatments did not affect BW during the experimental period. Antibody titers were increased in animals immunized against cGnRH-II and lGnRH-III (P < 0.001). Cross-reactivity of the antibodies to mammalian GnRH or between cGnRH-II and lGnRH-III was minimal. Concentrations of testosterone were maximal in control boars (treatment 3) and minimal in control barrows (treatment 4) and immunized pigs (treatment x week; P < 0.01). Immunized animals had concentrations of LH (P < 0.001) and FSH (treatment x week; P < 0.03) that were less than control barrows and similar to control boars. At the end of the experiment, intact (noncastrated) pigs were exsanguinated. Testes were removed immediately; Leydig cells were isolated and treated with 0, 1, or 10 ng/mL of LH. There was an LH x GnRH treatment effect on testosterone concentrations (P < 0.03), indicating that Leydig cells were sensitive to the immunization protocol and doses of LH. Taken together, these data suggest that immunization against GnRH isoforms decreased gonadotropin secretion compared with control barrows. Additionally, immunization against cGnRH-II and lGnRH-III reduced the ability of Leydig cells to respond to LH challenges.

Testosterone directly induces progesterone production and interacts with physiological concentrations of LH to increase granulosa cell progesterone production in laying hens ( Gallus domesticus)

Blocking testosterone action with immunization or with a specific antagonist blocks the preovulatory surge of progesterone and ovulation in laying hens. Thus, testosterone may stimulate progesterone production in a paracrine fashion within the ovary. To test this hypothesis, we evaluated the effects of testosterone and its interaction with LH on the production of progesterone by granulosa cells in culture. Hen granulosa cells obtained from preovulatory follicles were cultured in 96 well plates. The effects of testosterone (0–100 ng/ml) and/or LH (0–100 ng/ml) were evaluated. LH-stimulated progesterone production in a dose response manner up to 10 ng/ml ( p < 0.01). Testosterone, up to 10 ng/ml, increased progesterone production in a dose response manner in the absence of LH and at all doses of LH up to 1 ng/ml ( p < 0.001). However, at supraphysiological concentrations of LH (10 and 100 ng/ml) there was no further increase in progesterone production caused by testosterone ( p > 0.05). Finally, the addition of 2-hydroxyflutamide (0–1000 μg/ml) to hen granulosa cells cultured with 10 ng/ml of testosterone reduced progesterone production in a dose response manner ( p < 0.001). In conclusion, testosterone stimulates progesterone production in preovulatory follicle granulosa cells and interacts with physiological concentrations of LH to increase progesterone production. In addition, testosterone stimulation on granulosa cells is specific since the testosterone antagonist decreased testosterone stimulatory action.

Different Doses of Porcine Luteinizing Hormone in Precocious Puberty Induction in Gilts

The use of hormonal protocols in puberty induction and synchronization of oestrus has lead to an increase in the efficiency of replacement gilts. The aim of this study was to evaluate different doses of porcine LH in precocious puberty induction and oestrus synchronization in a homogeneous group of gilts. Sixty-seven homogeneous prepubertal gilts (Camborough 22) at 137 +/- 4 days of age and 87 +/- 7 kg were treated with three different hormonal protocols: T1--600 UI of equine chorionic gonadotrophin (eCG; Novormon) and after a 72-h period 5 mg of LH (Lutropin); T2--600 UI of eCG and a 72-h period 2.5 mg of LH; T3--600 UI of eCG and a 72-h period 1.25 mg of LH. The ovaries were examined at slaughter, on day 6 after the hormonal treatment. There were no statistical differences (p > 0.05) between the different LH doses in the percentage of the detected oestrus (T1 = 42.85%; T2 = 60.87%, T3 = 52.18%), oestrus duration (T1 = 41.44 +/- 16.30 h; T2 = 48.57 +/- 16.29 h, T3 = 39.33 +/- 11.42 h), number of corpora lutea (T1 = 9.61 +/- 5.43; T2 = 9.86 +/- 3.32, T3 = 8.13 +/- 5.52) and percentage of animals presenting ovarian cystic degeneration (T1 = 33.33%; T2 = 39.13%, T3 = 39.13%). The T2 (2.5 mg of LH) presented the lowest dispersion (p < 0.05) of the LH-ovulation interval (T1 = 37.17 +/- 4.07 h; T2 = 38.26 +/- 2.84 h; T3 = 36.25 +/- 5.69 h). The LH dose reduction to 2.50 and 1.25 mg presented equal results with the recommended dose of 5.0 mg, and could be used in the precocious induction of oestrus in gilts. The 2.5-mg LH dose showed the lowest dispersion of ovulation and it can be used in fixed-time artificial insemination programmes.