Development and characterization of in vitro self-assembled recombinant human follicle stimulating hormone originated from goat mammary epithelial cells.

Follicle stimulating hormone (FSH), composed of FSHα and FSHß subunits, is essential for female follicle development and male spermatogenesis. The recombinant human FSH (rhFSH) products on the market are mainly generated from mammalian cells and are expensive. Large animal mammary gland bioreactors are urgently needed to produce large amounts of rhFSH. However, there are currently no effective methods to prepare rhFSH by large animals mainly due to the fact that excessive accumulation of FSH might cause many adverse effects in animals. We herein report the development and characterization of functional self-assembled rhFSH produced in goat mammary epithelial cells (GMECs). FSHα and FSHß stably expressed in Chinese hamster ovary (CHO) cell lines were secreted into culture medium and well glycosylated. Importantly, FSHα and FSHß expressed apart were able to assemble into functional FSH. We next inserted human FSHα or FSHß gene separately into goat ß-Lactoglobulin locus in GMECs by CRISPR/Cas9. Inactive FSHα and FSHß subunits expressed from GMECs assembled into rhFSH as analyzed by His-tag pull down assay. Functional assessment of rhFSH by cAMP induction assay, mouse ovulation induction and rat ovarian weight gain experiments showed that the bioactivity of self-assembled rhFSH expressed by GMECs was comparable to that of Gonal-F both in vitro and in vivo. Our study demonstrated that FSHα and FSHß can be separately expressed and assembled into functional rhFSH, and provided the basis for future preparing FSH by goat mammary gland bioreactor with less health problems on the producing animals.

Conversion of TSH heterodimer to a single polypeptide chain increases bioactivity and longevity.

TSH is a dimeric glycoprotein hormone composed of a common α-subunit noncovalently linked to a hormone-specific ß-subunit. Previously, the TSH heterodimer was successfully converted to an active single-chain hormone by genetically fusing α and ß genes with [TSHß- carboxyl-terminal peptide (CTP)-α] or without (TSHß-α) the CTP of human chorionic gonadotropin ß-subunit as a linker. In the present study, TSH variants were expressed in Chinese hamster ovarian cells. The results indicated that TSHß-α single chain has the highest binding affinity to TSH receptor and the highest in vitro bioactivity. With regard to the in vivo bioactivity, all TSH variants increased the levels of T(4) in circulation after 2 and 4 h of treatment. However, the level of T(4) after treatment with TSH-wild type was significantly decreased after 6 and 8 h, compared with the levels after treatment with the other TSH variants. TSHß-α and TSHß-CTP-α single chains exhibited almost the same bioactivity after 8 h of treatment. Evaluating the half-life of TSH variants, TSHß-CTP-α single chain revealed the longest half-life in circulation, whereas TSH-wild type exhibited the shortest serum half-life. These findings indicate that TSH single-chain variants with or without CTP as a linker may display conformational structures that increase binding affinity and serum half-life, thereby, suggesting novel attitudes for engineering and constructing superagonists of TSH, which may be used for treating different conditions of defected thyroid gland activity. Other prominent potential clinical use of these variants is in a diagnostic test for metastasis and recurrence of thyroid cancer.

Effects of α and ß recombinant FSH (Gonal-F, Puregon) and progesterone upon human endometrial cell proliferation in-vitro: a preliminary study.

Endometrial proliferation or regeneration during menstrual cycle is regulated by sexual hormones. However, the effect of gonadotrophins on the endometrial cell growth remains obscure. Herein, we aimed to investigate the effects of r-FSH (Gonal-F, Puregon) and progesterone on the proliferation of human endometrial cells in-vitro. According as gonadotrophin concentrations, the follicular-phase endometrial cells were divided into six groups: (1) 0 (controls), (2) 1; (3) 10; (4) 100; (5) 1000; (6) 100,000 µIU/ml. The cell countings with microscopy and cell proliferation kit assay were used to assess the endometrial cell proliferations. In Gonal-F groups, the cell absorptions (%) after 24/48 h culture were: (1) 100/100; (2) 103.8/102.3; (3) 104.8/102.8; (4) 102.3/101.3; (5) 96.3/94.2; (6) 86.8/84.3. In Puregon groups, the cell absorptions were: (1) 100/100; (2) 102.8/101.9; (3) 103/102.3; (4) 103.9/103.5; (5) 102.9/102.4; (6) 103.7/103.2 (non-different). In progesterone groups, the cell absorptions were: (1) 100/100; (2) 99.1/101.9; (3) 83.5/80.4; (4) 80.7/82.4. Higher dosage of Gonal-F (100,000 µIU/ml) and progesterone (10, 100 µg/ml) appeared the significant inhibition upon endometrium. We conclude that lower dosages of Gonal-F, Puregon, and progesterone appear the non-significant influence upon endometrium. Higher dosage of Gonal-F (10,000 µIU/ml) and progesterone (10, 100 µg/ml), but not Puregon, might interfere with the endometrial proliferation during follicular phase.

Gonadal steroids and affective symptoms during in vitro fertilization: implication for reproductive mood disorders.

Gonadal steroids (GSs) have been associated with the onset of a number of reproductive-related mood disorders in women, in which fluctuating or unstable hormonal levels are postulated to act as the trigger for the destabilization of mood. There is, however, rather limited direct clinical evidence that can link rapidly changing GS levels with the induction of mood symptoms. We aimed to study the effect of controlled and rapid GS fluctuations on mood in an in vivo model. Women undergoing in vitro fertilization (n=108) were assessed for depression and anxiety levels on 3 time points: during a low estradiol and progesterone baseline, during a gonadotropin stimulated estradiol-dominant phase, and after embryo transfer, during a progesterone-dominant low estrogen phase. Plasma levels for estrogen and progesterone were drawn on these time points. Symptoms of depression and anxiety significantly increased from baseline to the high estradiol levels but were not correlated with estrogen. The sharp drop from high estradiol levels at the estradiol-dominant phase to low levels at the progesterone-dominant phase was significantly correlated with rising depression scores. The rise in progesterone levels from low levels at the estradiol-dominant phase to high levels at the progesterone-dominant phase was significantly and inversely correlated with depression scores. This study suggests that the mechanism underlying the role of estrogen in reproductive-related mood disorders involves an abrupt and precipitous drop in its plasma level that can precipitate negative mood states. This finding has implications on the treatment of GS-related mood disorders.

[Differing response to GnRH antagonists in cycles of ovarian hyperstimulation plus intrauterine insemination]. / Diferencia de respuesta a los antagonistas de GnRH en ciclos de hiperestimulación ovárica más inseminación intrauterina.

OBJECTIVE: To compare two flexible protocols of GnRHant in OH plus IUI vs a control group without GnRHant. MATERIALS AND METHODS: Randomized controlled trial 90 infertile patients were analyzed in 116 cycles of IUI. Cycles were randomized in 3 groups; group 1: started GnRHant when the leading follicle reached 14mm, group 2: when it reached 16mm and group 3: without GnRHant hormonal determinations were done during OH. Main outcomes were: premature LH raise incidence, premature luteinization (PL) and pregnancy rate per cycle. RESULTS: Premature LH rise incidence was 2.6% (3 cycles) and PL 6% (7 cycles). Group 1 didn't present cycles with LH rise or PL, groups 2 and 3 presented LH rise in 3.1% and 1.8% and PL in 12.5% and 5.4% respectively. Pregnancy rate with GnRHant was 16.4% (95% IC 8.1-28.1) vs. 7.2% (95% le 2.0-17.5%) without GnRHant (group 3) (p = 0.16): groups 1 and 2 represented a pregnancy rate of 20.6% (95% IC 7.9-39.7) and 12.5% (95% IC 3.5-28.9%) respectively (p = 0.49). Mature follicles number reached meaning difference between all groups (p = 0.04) specially between groups 1 and 2 (p = 0.02). CONCLUSIONS: A trend to elevate pregnancy rates was observed with GnRHant specially with when it was started when leading follicle reached 14 mm (p > 0.05). Starting GnRHant with 16 mm was not totally usefully to prevent PL.

Stimulation of spermatogenesis with recombinant human follicle-stimulating hormone (follitropin alfa; GONAL-f): long-term treatment in azoospermic men with hypogonadotropic hypogonadism.

OBJECTIVE: To demonstrate the efficacy and safety of follitropin alfa administered with hCG on spermatogenesis in adult male hypogonadotropic hypogonadism (HH) patients. DESIGN: Phase III, multicenter, open-label, noncomparative. SETTING: Seven US medical centers. PATIENT(S): A total of 36 adult males with severe HH. INTERVENTION(S): A total of 1,000 U hCG on alternate days for 3 to 6 months, with dose adjustments after 2 months, if necessary, to normalize T levels, followed by follitropin alfa 150 U and hCG on the same alternate days for 18 months, with dose adjustments as necessary. MAIN OUTCOME MEASURE(S): Proportion of patients with sperm density > or =1.5 x 10(6)/mL. Pubertal advancement and long-term safety and tolerability were also evaluated. RESULT(S): In total, 22 of 29 patients (75.9%) who received > or =1 dose of follitropin alfa and 20 of 25 patients (80%) who completed 18 months of hCG + follitropin alfa treatments achieved a sperm concentration > or =1.5 x 10(6)/mL. A sperm concentration >20 x 10(6)/mL was achieved by 8 of 29 men (27.5%). Median sperm concentration at 18 months was 5.2 x 10(6)/mL. Pubertal development continued during the study, and testis volumes increased. Five clinical pregnancies were achieved. Acne (52% of patients) was the most common side effect, and gynecomastia was reported in 10% of patients. CONCLUSION(S): Long-term treatment of azoospermic HH men using follitropin alfa and hCG is effective for stimulating spermatogenesis and is well-tolerated.

Assessment of the biopotency of follitropin alfa and lutropin alfa combined in one injection: a comparative trial in Sprague-Dawley rats.

BACKGROUND: The current study was designed to determine if follitropin alfa (recombinant human follicle-stimulating hormone; r-hFSH) and lutropin alfa (recombinant human luteinizing hormone; r-hLH) biopotencies were unchanged by reconstituting in sterile water for injection and mixing prior to injection. METHODS: The biopotencies of r-hFSH and r-hLH were determined following injection of female Sprague-Dawley rats with a mixture of follitropin alfa revised formulation female (RFF) and lutropin alfa (1:1, r-hFSH:r-hLH). Biopotencies of follitropin alfa RFF and lutropin alfa were measured using ovarian weight and ascorbic acid depletion assays, respectively, and compared with a reference standard. Stock mixtures of follitropin alfa RFF and lutropin alfa (1:1) were prepared within 1 h prior to each respective assay's injection and stored at 6 +/- 2 degrees C. Separate low dose (follitropin alfa RFF 1.5 IU/rat, lutropin alfa 2 IU/rat) and high dose (follitropin alfa RFF 3 IU/rat, lutropin alfa 8 IU/rat) treatments were prepared from stock mixtures or individual solutions by diluting with 0.22% bovine serum albumin saline solution and injected within 1 h of preparation. The main outcome measures were ovarian weight and ovarian ascorbic acid depletion. RESULTS: FSH bioactivities were similar (p > 0.10) between the individual follitropin alfa RFF test solution (84.2 IU) and follitropin alfa RFF/lutropin alfa (87.6 IU) mixtures prepared within 1 h of injection and stored at 6 +/- 2 degrees C. LH bioactivities were similar (p > 0.10) between lutropin alfa (94.7 IU) test solution and lutropin alfa/follitropin alfa RFF (85.3 IU) mixtures prepared within 1 h of injection and stored at 6 +/- 2 degrees C for not more than 1 h prior to injection. CONCLUSION: Mixing follitropin alfa RFF and lutropin alfa did not alter the bioactivity of either FSH or LH.

Lutropin alfa.

Lutropin alfa is the first and only recombinant human form of luteinizing hormone (LH) developed for use in the stimulation of follicular development. Dose-finding studies revealed a significant dose-dependent increase in the rate of optimal follicular development among women with hypogonadotropic hypogonadism and profound LH deficiency (<1.2 IU/L) who received subcutaneous lutropin alfa 0-225 IU/day plus follitropin alfa. Similarly, in a double-blind, randomized study, the rate of optimal follicular development was significantly higher in women with hypogonadotropic hypogonadism and profound LH deficiency receiving subcutaneous lutropin alfa 75 IU/day plus follitropin alfa than in those receiving placebo plus follitropin alfa. Lutropin alfa with follitropin alfa may also be of benefit in certain subgroups of normogonadotropic women (e.g. those with an inadequate response to prior follitropin alfa monotherapy, those aged >or=35 years, and those with profound LH downregulation or who required excessive exogenous follitropin alfa). However, one study in older women (>or=35 years) did not show any advantage of lutropin alfa supplementation. Once-daily subcutaneous lutropin alfa was generally well tolerated in hypogonadotropic hypogonadal women, with the majority of adverse events being of mild to moderate severity.

Production of recombinant channel catfish (Ictalurus punctatus) FSH and LH in S2 Drosophila cell line and an indication of their different actions.

Due to the lack of purified, native gonadotropins (GtH) for almost all species of fish, we designed a system for the production of recombinant bioactive luteinizing hormone (LH) and follicle stimulating hormone (FSH) using the channel catfish (Ictalurus punctatus) as a model animal. The strategy was to produce the three subunits composing FSH and LH, i.e. the common alpha-subunit (alpha-glycoprotein hormone (alpha-GP)), beta-FSH, and beta-LH subunit, individually in stable recombinant insect cells (S2) with C-terminal His-tag. This expression system was also used to co-express the alpha-subunit without the His-tag with each of the His-tagged beta-subunits. The recombinant S2 cells were capable of secreting FSH and LH heterodimers and alpha-GP in abundance; however, expression of the individual beta-subunits was much less successful. The recombinant GtHs were partially purified from the cell medium by immobilized metal affinity chromatography to ~15% purity with a yield of 7 and 4 mg per liter of medium for FSH and LH respectively. These recombinant GtHs activated their receptors in vitro, enhanced estrogen secretion, up-regulated several steroidogenic enzyme genes in channel catfish ovarian follicles, and increased androgen secretion from African catfish testis. Interestingly, the FSH and LH dose-response curves for each of these biological activities clearly demonstrate differences in their cellular action and physiological roles. This expression system may be an important development for the production of species-specific GtHs so that FSH- and LH-specific mechanisms of actions within the reproductive endocrine processes can finally be examined with homologous, albeit recombinant, hormones.

A comparison between follitropin alpha filled by mass and follitropin alpha filled by bioassay in the same egg donors.

This study assessed the ovarian stimulation characteristics of recombinant follitropin alpha filled by mass (rFSH-fbm) versus recombinant follitropin alpha filled by conventional bioassay (rFSH-bio) in the same egg donor patients. Eleven egg donors, who had two ovarian stimulation cycles for oocyte retrieval (total of 23 cycles), one with rFSH-bio (Gonal-f Multidose) and the second one with rFSH-fbm (Gonal-f RFF), were evaluated. The protocol of ovarian stimulation was exactly the same in both cycles, consisting of GnRH suppression (luteal phase) followed by exclusive stimulation with rFSH. Despite no differences in the number of days of rFSH treatment and in the total dosage of rFSH administered, the number of follicles >14 mm and the number of oocytes retrieved were significantly higher in the rFSH-fbm group (P = 0.01 and 0.04 respectively). The mean oestradiol peak values showed a trend in favour of rFSH-fbm (3123 versus 2405 pg/ml respectively). These results suggest that the consistency in dosing provided by follitropin alpha filled by mass as opposed to follitropin alpha filled by bioassay offers added value for the ovarian stimulation of oocyte donor patients.

Effects of different human chorionic gonadotrophin preparations on trophoblast differentiation.

Recent evidence from the literature suggested that hCG preparations purified from urine of pregnant women, which are widely used in in vitro studies and IVF programs, may contain contaminants such as EGF. To determine the putative biological effects of the contaminating growth factor, we here investigated distinct trophoblast differentiation processes in the presence of various hCG compounds. Western blot analyses indicated that treatment of trophoblastic SGHPL-5 cells and purified term trophoblasts with potentially EGF-contaminated hCG (hCG-A) resulted in auto-phosphorylation of the EGF receptor at tyrosine 1173 whereas supplementation of another urine-purified hCG preparation (hCG-B), recombinant holo-hCG or recombinant alphahCG had no effects. Phosphorylation was specifically blocked by the EGF receptor inhibitor PD153035. Urinary hCG-A was most effective in promoting invasion of SGHPL-5 cells through Matrigel-coated transwells, but increased invasiveness was also observed in the presence of hCG-B or recombinant holo-hCG. Similarly, the extent of syncytialisation of term trophoblasts, quantitated by nuclei in desmoplakin-negative areas, was highest upon addition of hCG-A or recombinant EGF as a control. PD153035 reduced invasion and fusion of trophoblasts supplemented with hCG-A, but did not diminish the effects provoked by hCG-B. In conclusion, the data suggest that the EGF contamination of hCG considerably affects trophoblast function. Experiments using EGF-free hCG preparations demonstrate that the hormone increases trophoblast invasion and syncytialisation.

Single-chain, triple-domain gonadotropin analogs with disulfide bond mutations in the alpha-subunit elicit dual follitropin and lutropin activities in vivo.

The human glycoprotein hormones chorionic gonadotropin (CG), TSH, LH, and FSH are heterodimers composed of a common alpha-subunit and a hormone-specific beta-subunit. The subunits assemble noncovalently early in the secretory pathway. LH and FSH are synthesized in the same cell (pituitary gonadotrophs), and several of the alpha-subunit sequences required for association with either beta-subunit are different. Nevertheless, no ternary complexes are observed for LH and FSH in vivo, i.e. both beta-subunits assembled with a single alpha-subunit. To address whether the alpha-subunit can interact with more than one beta-subunit simultaneously, we genetically linked the FSHbeta- and CGbeta-subunit genes to the common alpha-subunit, resulting in a single-chain protein that exhibited both activities in vitro. These studies also indicated that the bifunctional triple-domain variant (FSHbeta-CGbeta-alpha), is secreted as two distinct bioactive populations each corresponding to a single activity, and each bearing the heterodimer-like contacts. Although the data are consistent with the known secretion events of gonadotropins from the pituitary, we could not exclude the possibility whether transient intermediates are generated in vivo in which the alpha-subunit shuttles between the two beta-subunits during early stages of accumulation in the endoplasmic reticulum. Therefore, constructs were engineered that would direct the synthesis of single-chain proteins completely devoid of heterodimer-like interactions but elicit both LH and FSH actions. These triple-domain, single-chain chimeras contain the FSHbeta- and CGbeta-subunits and an alpha-subunit with cystine bond mutations (cys10-60 or cys32-84), which are known to prevent heterodimer formation. Here we show that, despite disrupting the intersubunit interactions between the alpha- and both CGbeta- and FSHbeta-subunits, these mutated analogs exhibit both activities in vivo comparable to nonmutated triple-domain single chain. Such responses occurred despite the absence of quaternary contacts due to the disrupted bonds in the alpha-subunit. Thus, gonadotropin heterodimer assembly is critical for intracellular events, e.g. hormone-specific posttranslational modifications, but when heterodimers are present in the circulation, the alpha/beta-contacts are not a prerequisite for receptor recognition.

Expression of Pin1, a peptidyl-prolyl isomerase, in the ovaries of eCG/hCG-treated immature female mice.

Protein phosphorylation on certain serine or threonine residues preceding proline (Ser/Thr-Pro) is a pivotal signaling mechanism in diverse cellular processes. Pin1 is a highly conserved enzyme that isomerizes only the phosphorylated Ser/Thr-Pro bonds in certain proteins, thereby inducing conformational changes. Although much protein is phosphorylated in the ovary, the role of Pin1 in the ovary is still unknown. The purpose of this study is to investigate the effects of gonadotropins on protein and mRNA expression of Pin1 in mice ovaries. Quantitative PCR analysis showed that the expression of Pin1 mRNA significantly increased in the ovaries of equine chorionic gonadotropin (eCG)-treated mice compared with those of untreated mice (P<0.05). However, human chorionic gonadotropin (hCG) attenuated the expression of Pin1 mRNA increased by eCG. The protein level of Pin1 showed the same tendency as the expression of mRNA. The mRNA expression of E2F transcription factor, which controlled the expression of Pin1, was significantly decreased in the eCG-treated ovaries compared with the controls (P<0.05). These observations suggest that gonadotropins may regulate the expression of Pin1 without E2F transcription factor, indicating that Pin1 might be an important factor for protein signal transduction during follicular development.

CR6-interacting factor 1 interacts with orphan nuclear receptor Nur77 and inhibits its transactivation.

CR6-interacting factor 1 (CRIF1) was recently identified as a nuclear protein that interacts with the Gadd45 (growth arrest and DNA damage inducible 45) family of proteins and participates in the regulation of the G1/S phase of the cell cycle. However, the nuclear action of CRIF1 is largely unknown. In this study, we demonstrate that CRIF1 acts as a novel coregulator of transactivation of the orphan nuclear receptor Nur77. Both in vitro and in vivo studies show that CRIF1 interacts with Nur77 via the Nur77 AB domain and that it dramatically inhibits the AB domain-mediated transactivation of Nur77. Transient transfection assays demonstrate that CRIF1 inhibits steroid receptor coactivator-2-mediated Nur77 transactivation, and silencing of endogenous CRIF1 by small interfering RNA relieves this repression. CRIF1 possesses intrinsic repressor activities that are not affected by the histone deacetylase inhibitor Trichostatin A. In addition, overexpression of CRIF1 inhibits TSH/protein kinase A-induced Nur-responsive element promoter activity. CRIF1 inhibited Nur77-dependent induction of E2F1 promoter activity, mRNA expression, and Nur77-mediated G1/S progression in cell cycle. These results suggest that CRIF1 acts as a repressor of the orphan nuclear receptor Nur77 by inhibiting AB domain-mediated transcriptional activity.

Transexualismo / Transsexualism

La demanda para la cirugía de reasignación de sexo en pacientes transexuales ha aumentado considerablemente. Los transexuales desean vivir permanentemente como miembros del sexo opuesto. Este deseo, acompañado de un profundo rechazo de sus propios caracteres sexuales primarios y secundarios, es absoluto, sofocante e inmodificable. Como consecuencia de este comportamiento psicológico, la persona transexual busca realizarse corrigiendo la apariencia sexual de su cuerpo mediante métodos quirúrgicos y farmacológicos. En 1979 se creó la Harry Benjamin International Gender Dysphoria Association, que recomendó unas directrices asistenciales que sirvieran de base para atender los trastornos de identidad de género. El tratamiento hormonal representa un importante papel en el proceso, que debe eliminar, idealmente, los caracteres sexuales secundarios del sexo original e inducir los del sexo opuesto tan rápida y completamente como sea posible. Hay tendencia a tomar hormonas cuanto antes y a maximizar las dosis, usando diversas pautas aprendidas de la experiencia de otros transexuales. Esta forma de automedicación con esteroides sexuales incrementa el riesgo de efectos adversos. La aproximación a este trastorno es compleja. Para atenderlo es necesario un equipo multidisciplinario en el marco de la medicina pública, dentro del Sistema Nacional de Salud. Los beneficios son evidentes: mejora la calidad de vida del paciente y su nivel de satisfacción y, desde el punto de vista médico, son importantes los beneficios del tratamiento hormonal y el éxito del tratamiento quirúrgico a corto y a largo plazo (AU)

Stimulating effect of glycoprotein hormone free alpha-subunit and daily gonadotropin releasing hormone treatment on prolactin release from 50-day ovine foetal pituitary explants.

The aim of our study was to determine whether free alpha of glycoprotein hormones (free alpha) plays a role in lactotroph function during early pituitary development in the sheep foetus. Detection and quantification of free alpha, luteinzing hormone beta-subunit (LHbeta) and prolactin immunolabelling were determined by immunocytochemistry at days 32, 37, 42, 50 and 63 of gestation. Free alpha- and LHbeta-containing cells were first detected in the ovine foetal pituitary gland on day 37 of gestation, while prolactin-containing cells were first identified on day 42. Analysis of serial sections suggested that free alpha immunoreactive cells were also LHbeta-positive, indicating that free alpha was mainly synthesized by gonadotrophs. In early foetal stages, free alpha occurred in the antero-medio ventral region of the pituitary gland, whereas prolactin-containing cells were more dorsally and more caudally localized. The free alpha-, LHbeta- and prolactin-immunostained area increased markedly between days 50 and 63 of gestation. To evaluate a possible functional relationship between gonadotrophs and lactotrophs, the effects of free alpha or gonadotropin releasing hormone (GnRH) on prolactin release were assayed. Chronic treatment of pituitary explants from male and female 42-day-old ovine foetuses for 8 days with 10-9 or 10-7 M ovine free alpha did not affect prolactin release. By contrast, free alpha administration on pituitary explants from male and female 50-day-old foetuses resulted in enhanced prolactin release. At this age, a daily (2 h per day) treatment with 10-8 M GnRH had similar stimulatory effect to free alpha whereas a 'first day' treatment (24 h on the first day) reduced prolactin release throughout the culture in males and had no effect in females. These results indicate that, despite early detection of free alpha at day 37 in the ovine foetal pituitary, its stimulatory effect on prolactin release occurs from day 50 of gestation, corresponding to the first period of lactotroph development in vivo. A daily treatment with GnRH mimics the effect of free alpha on prolactin release.

Biological activity of single chain chorionic gonadotropin, hCGalphabeta, is decreased upon deletion of five carboxyl terminal amino acids of the alpha subunit without affecting its receptor binding.

The strategy of translationally fusing the two subunits of human chorionic gonadotropin (hCG) has been used to produce recombinant single chain hCG in which the C-terminus of the alpha subunit is fused to the N-terminus beta without any linker using Pichia pastoris expression system. The Pichia-expressed hCGalphabeta (phCGalphabeta) attained an overall conformation similar to that of hCG, and could bind to the receptor and elicit biological response, suggesting that receptor binding and signal transduction can take place even with a molecule having blocked the C-terminus of the alpha subunit. The carboxyl terminal of the alpha subunit has been shown to be involved in hormone binding and signal transduction of all the heterodimeric glycoprotein hormones. However, deletion of five amino acids from the C-terminus of the alpha subunit in the single chain hCG did not alter the overall conformation of the fusion molecule and its receptor binding ability, but led to a significant reduction in its ability to elicit biological response. These data show that these five amino acids at the C-terminus of the alpha subunit in the single chain hCG are not absolutely essential for attaining a conformation required for receptor binding, but are essential for obtaining a full biological response.