A human-based assisted reproduction protocol for the menstruating spiny mouse, Acomys cahirinus.

The Egyptian or Common spiny mouse (A. cahirinus) is the first rodent species to show human-like menstruation and spontaneous decidualisation. We consider from these, and its other, human-like characteristics that this species will be a more useful and appropriate small animal model for human reproductive studies. Based on this, there is a need to develop specific laboratory-based assisted reproduction protocols including superovulation, in-vitro fertilisation, embryo cryopreservation and transfer to expand and make this model more relevant. Because standard rodent superovulation has not been successful in the spiny mouse, we have selected to test a human protocol. Female spiny mice will receive a subcutaneous GnRH agonist implant and be allowed to recover. Menstrual cycle lengths will then be allowed to stabilize prior to ovarian stimulation. After recovery, females will be injected IP once a day for 4 days with a FSH analogue, to induce follicular growth, and on day 5 will be injected IP with a hCG analogue to trigger ovulation. Females will either be culled 36hrs after trigger to collect oocytes or immediately paired with a stud male and two cell embryos collected 48hrs later. Mature oocytes will be inseminated using fresh spiny mouse spermatozoa and all in-vitro grown and in-vivo collected two cell embryos will be cryopreserved using methods developed in a close spiny mouse relative, the Mongolian gerbil. For embryo transfer, vitrified embryos will be rapidly warmed and non-surgically transferred to surrogate mice. Surrogates will be monitored until pregnancy is apparent (roughly 30 days) and then left undisturbed until birth, 38-40 days after transfer. By successfully developing robust assisted reproduction protocols in A. cahirinus we will be able to use this rodent as a more effective model for human reproduction.

High cGMP and low PDE3A activity are associated with oocyte meiotic incompetence.

Resumption of meiosis in mammalian oocytes, defined as oocyte maturation, is stimulated by luteinizing hormone (LH). Fully grown oocytes can also mature spontaneously, upon their release from the ovarian follicle. However, growing oocytes fail to resume meiosis in vitro and the mechanism underlying their meiotic incompetence is unknown. It is commonly accepted that a drop in intraoocyte cyclic guanosine monophosphate (cGMP) resulting in the elevated activity of the oocyte-specific PDE3A leads to a decrease in cAMP content, essential for reinitiation of meiosis. We explored the regulation of these cyclic nucleotides and their degrading PDE3A in growing oocytes. Our research addressed the LH-induced rather than spontaneous oocyte maturation. We examined 16-21 as compared to 25-day-old, PMSG-primed rats, treated with the LH analog, hCG. The effect of LH was also examined ex vivo, in isolated ovarian follicles. We found that hCG failed to induce oocyte maturation and ovulation in the younger animals and that ovulation-associated genes were not upregulated in response to this gonadotropin. Furthemore, the drop of intraoocyte cGMP and cAMP observed in fully grown oocytes upon exposure of the ovary to LH, was not detected in growing oocytes. Interestingly, whereas the global expression of PDE3A in growing and fully grown oocytes is similar, a significantly lower activity of this enzyme was determined in growing oocytes. Our findings show that meiotic incompetence is associated with a relatively high oocyte cGMP concentration and a low activity of PDE3A, which in follicle-enclosed oocytes may represent the failure of the somatic follicle cells to respond to LH.

Rerouting of a follicle-stimulating hormone analog to the regulated secretory pathway.

LH and FSH are produced by the same gonadotrope cells of the anterior pituitary but differ in their mode of secretion. This coordinated secretion of LH and FSH is essential for normal follicular development and ovulation in females and for spermatogenesis in males. The structural signals encoded in the LH and FSH subunits that govern the intracellular sorting of LH through the regulated secretory pathway and FSH through the constitutive pathway are largely unknown. Our laboratory recently identified the seven amino acid carboxy tail of LH beta as a sorting signal for LH in GH(3) cells. Here we compared the morphological features of GH(3) cells expressing an FSH analog containing the heptapeptide (FL7AA) with wild-type FSH using confocal microscopy. These experiments were performed to develop a rerouting model for examining structure-function links between secretion pathways of FSH/LH and their biological action. Both FSH- and LH-expressing cells exhibit a fluorescence pattern of randomly dispersed cytoplasmic puncta. FL7AA expressing cells have more intracellular accumulation compared with wild-type FSH and display a unique halo pattern of fluorescence near the plasma membrane. Such a pattern was not observed in cells expressing FSH or LH. Our results demonstrate that this FSH analog containing the carboxy heptapeptide of LH beta is rerouted to the regulated secretory pathway in GH(3) cells. This rerouted gonadotropin provides a unique model to study the trafficking, regulation, and function of LH and FSH.

Coactivation of estrogen receptor beta by gonadotropin-induced cofactor GIOT-4.

Estrogen exerts its diverse effects through two subtypes of estrogen receptors (ER), ERalpha and ERbeta. Each subtype has its own distinct function and expression pattern in its target tissues. Little, however, is known about the transcriptional regulatory mechanism of ERbeta in the major ERbeta-expressing tissues. Using biochemical methods, we identified and described a novel ERbeta coactivator. This protein, designated GIOT-4, was biochemically purified from 293F cells. It coactivated ERbeta in ovarian granulosa cells. GIOT-4 expression was induced by stimulation with follicle-stimulating hormone (FSH). GIOT-4 recruited an SWI/SNF-type complex in a ligand-independent manner to ERbeta as an ER subtype-specific physical bridging factor and induced subsequent histone modifications in the ERbeta target gene promoters in a human ovarian granulosa cell line (KGN). Indeed, two ERbeta-specific target genes were upregulated by FSH at a specific stage of a normal ovulatory cycle in intact mice. These findings imply the presence of a novel regulatory convergence between the gonadotropin signaling cascade and ERbeta-mediated transcription in the ovary.

Among patients treated with FSH and GnRH analogues for in vitro fertilization, is the addition of recombinant LH associated with the probability of live birth? A systematic review and meta-analysis.

The aim of this systematic review and meta-analysis was to assess whether the addition of recombinant luteinizing hormone (LH) increases live birth rate, among patients treated with follicle stimulating hormone (FSH) and gonadotrophin-releasing hormone (GnRH) analogues for in vitro fertilization (IVF). Eligible studies were randomized controlled trials (RCTs) answering the research question that contained sufficient information to allow ascertainment of whether randomization was true and whether equality was present between the groups compared, regarding baseline demographic characteristics, gonadotrophin stimulation protocol, number of embryos transferred and luteal phase support administered. A literature search identified seven RCTs (701 patients) that provided the information of interest, among which five reported agonist and two antagonist cycles. The reported outcome measure, clinical pregnancy, was converted to live birth using published data in one study. No significant difference in the probability of live birth was present with or without rLH addition to FSH (odds ratio [OR]: 0.92, 95% confidence interval (CI): 0.65-1.31; P = 0.65). This finding remained stable in subgroup analyses that ordered the studies by dose of rLH added, the type of analogue used to inhibit premature LH surge, the time rLH was added during the follicular phase, the age of patients analysed, the presence of allocation concealment and by the way the information on live birth was retrieved. In conclusion, the available evidence does not support the hypothesis that the addition of recombinant LH increases the live birth rate in patients treated with FSH and GnRH analogues for IVF.

Cysteine residues in a synthetic peptide corresponding to human follicle-stimulating hormone beta-subunit receptor-binding domain 81-95 [hFSH-beta-(81-95)] modulate the in vivo effects of hFSH-beta-(81-95) on the mouse estrous cycle.

We have previously reported that synthetic peptide amides corresponding to subdomains of the human FSH 3-subunit, hFSH-beta-(33--53) and hFSH-beta-(81--95), interact with the external domain of the FSH receptor in two in vitro model systems. Consistent with these in vitro observations, we found that intraperitoneal (i.p.) administration of each of these peptides prolonged vaginal estrus in normally cycling mice in vivo. Both hFSH-beta-(33--53) and hFSH-beta-(81--95) contain cysteine (Cys) residues with free sulfhydryl groups of potential significance in receptor interactions. To assess the possible involvement of these groups in the in vivo effects of hFSH-beta-(33--53) and hFSH-beta-(81--95), synthetic peptide analogs were prepared in which all Cys residues were replaced with serine (Ser). In the present study, we demonstrate that the in vivo effect of hFSH-beta-(33--53) on the mouse estrous cycle, extension of vaginal estrus, was not changed by substitution of Cys-51 with Ser. In contrast, mice receiving the Ser-substituted analog of hFSH-beta-(81--95) had normal estrus stages, but were arrested in diestrus. hFSH-beta-(33--53)-(81--95), a linear peptide encompassing both domains, also prolonged vaginal estrus. The Ser-substituted analog of this peptide, however, prolonged vaginal estrus in some of the mice tested and induced cycle arrest at diestrus in others. hFSH-beta-(90--95), the active subdomain at the C-terminus of hFSH-beta-(81--95), extended vaginal estrus, but diestrus stages were of normal duration. Its Ser-substituted analog, however, prolonged the estrus stage of the majority of mice treated, but induced diestrus arrest in some. The differing responses to these peptides are presumably due to interactions of the synthetic peptides with different regions of the FSH receptor. This further suggests that one consequence of ligand interaction with multiple receptor binding domains may be variable effects on ovarian function, and that Cys to Ser analogs may have value in the design of a novel class of synthetic peptides capable of fertility regulation and control.

Superovulation in cattle: effect of FSH type and method of administration on follicular growth, ovulatory response and endocrine patterns.

Although different FSH preparations and injection regimens are used to superovulate cattle, the optimum treatment regimen and blood concentrations of FSH to induce effective superovulatory responses are currently not known. The current objective was to evaluate the pattern of follicular growth, oestradiol-17 beta(E2) concentrations and yield of embryos in heifers following superovulation with two different pFSH preparations reportedly differing in LH content. In experiment 1, 90 synchronised heifers were superovulated at mid-cycle using a 2 x 2 factorial design comparing Folltropin (Vetrepharm; low LH) with Pluset (Serovet; FSH:LH ratio 1:1) administered either as a single or multiple (8 for Folltropin, MF and 10 for Pluset, MP) injections. Animals were inseminated during oestrus which was induced with prostaglandin F2 alpha analogue and embryos were recovered 7 days later. Overall, Pluset treatments compared with Folltropin resulted in more ovulations and unfertilized or degenerate embryos (P < 0.05). Multiple injections resulted in more (P < 0.05) freezable (MF = 55 +/- 1.2; MP = 3.8 +/- 1.0) and transferable embryos (MF = 2.68 +/- 0.9; MP = 2.71 +/- 0.9) than single injections (SF = 2.2 +/- 0.5 and 1.0 +/- 0.3 respectively; SP = 2.6 +/- 0.8 and 1.3 +/- 0.4 respectively); there was also a higher (P < 0.05) percentage embryo recovery rate. In two subsequent experiments, animals (n = 17) were superovulated with either single or multiple injections of Folltropin or Pluset as described and blood samples were collected and analysed for E2 concentrations. Ovarian scanning was carried out until 72 h after the first FSH injection, to count medium (5-9 mm) and large (> or = 10 mm) follicles. Heifers treated with SP had higher E2 concentrations in comparison with heifers treated with SF at 18, 36-48 and 84-96 h after the FSH injection. There were no differences in E2 concentrations in heifers treated with MF or MP treatments. Heifers treated with SP had greater numbers of follicles compared to SF treated heifers (21.0 +/- 3.1 vs 13.9 +/- 2.2; P = 0.089) on the third day after FSH injection. There were no differences between the numbers of medium and large follicles in heifers treated with MF or MP at any time throughout the experimental period. These data indicate that a single injection of Folltropin or Pluset can result in multiple ovulations and that the E2 profiles are different following single injections of either Folltropin or Pluset.

Regulation of insulin-like growth factor I biosynthesis in porcine granulosa cells.

In the present studies we examined the regulation of insulin-like growth factor I (IGF-I) expression in porcine granulosa cells in vitro. Using Northern analysis and ribonuclease protection assays with exon-specific probes, we identified the IGF-I messenger RNA (mRNA) transcripts present in these cells under basal and hormone-stimulated conditions. We also assessed changes in secreted IGF-I using Western blots and correlated the change in protein secretion after hormone treatment with changes in mRNA levels. By analogy to the human IGF-I gene and its transcription, two major transcripts of approximately 1 and 7.5 kilobases, seen in freshly isolated granulosa cells and follicle wall and in single passaged granulosa (MDGp1) cells, most likely correspond to IGF-IA. Minor transcripts of 3-4 kilobases, which appeared after FSH or forskolin treatments or in control cells after long exposure of the autoradiographs, were attributed to incompletely processed RNA precursors. Ribonuclease protection assay analysis using probes to detect alternative use of exon 5 or exon 6 indicated that most, if not all, of the transcripts contained only exon 6 sequence (IGF-IA). Both class 1 and class 2 transcripts were identified using exon 1- and exon 2-specific probes, respectively. GH increased steady state levels of IGF-I mRNA 3-fold, FSH increased it approximately 10-fold, and forskolin maximally increased it 12- to 15-fold. Estradiol had no effect alone or in combination with the other treatments. All treatments that increased IGF-I mRNA coordinately increased both class 1 and class 2 transcripts, with the increase in class 1 greater than that in class 2. Multiple forms of IGF-I protein were seen under basal conditions and after hormone treatment. These were identified based on mRNA analysis and biochemical methods as both glycosylated and nonglycosylated IGF-IA prohormone, incompletely processed forms of prohormone, and the mature peptide. Changes in the levels of total protein were similar to the changes in mRNA (GH, 3-fold; FSH and forskolin, 10- to 20-fold). All forms of the protein changed coordinately, suggesting that these hormones had no major effect on the intracellular processing mechanism. IGF-binding protein-3 was able to bind to all IGF-I forms. These data conclusively demonstrate FSH and GH induction of ovarian IGF-I. The porcine granulosa cell culture system used in these studies should be an excellent system for studying the hormonal regulation of IGF-I expression.

Enhanced stimulation of follicle maturation and ovulatory potential by long acting follicle-stimulating hormone agonists with extended carboxyl-terminal peptides.

The induction of granulosa cell differentiation and follicle maturation is dependent upon the stimulatory actions of FSH. Our recent studies used recombinant DNA technology to fuse the carboxyl-terminal peptide (CTP) of hCG beta-subunit to the carboxyl-terminus of the FSH beta-subunit. The resulting FSH analog has identical in vitro receptor-binding and biological activities as wild-type FSH (WT-FSH), but an increased circulating half-life. The present studies examined further the ability of FSH with one (FSH-CTP1) or two (FSH-CTP2) appended CTPs to promote granulosa cell differentiation and follicle ovulatory potential. WT-FSH, FSH-CTP1, and FSH-CTP2 were produced from Chinese hamster ovary cells transfected with the common alpha-subunit and respective beta-subunit. Hormone concentrations were quantitated by RIA, and relative levels confirmed by radioligand receptor assay. Both FSH-CTP1 and FSH-CTP2 retained full FSH receptor-binding activity, but did not bind LH receptors. To compare in vivo bioactivity, immature estrogen-primed female rats received ip injections of FSH or the agonists at 0 and 24 h. At 48 h, substantial stimulation (up to 2.5-fold) of ovarian weight was induced by 1.0 and 3.0 IU/day FSH-CTP1 or FSH-CTP2, whereas a higher dose (10 IU/day) of WT-FSH was required for an 1.8-fold stimulation. Although the in vivo potencies of FSH-CTP1 and FSH-CTP2 were similar, FSH-CTPs were about 10-fold more potent than WT-FSH in inducing granulosa cell aromatase activity and LH receptors. We further reduced the frequency of hormone administration. Increasing doses (1-10 IU) of a single ip injection of FSH-CTP1 resulted in dose-dependent increases in granulosa cell aromatase activity and LH receptor content 48 h later. Although a single injection (10 IU) of WT-FSH had no effect, the same total dose of WT-FSH administered as four 2.5-IU injections 12 h apart was effective. To test the ovulatory potential of ovarian follicles, rats received a single injection of FSH-CTP1, followed 52 h later by 5 IU hCG to induce ovulation. Although hCG did not induce ovulation in females receiving a single dose (10 IU) of WT-FSH, 20 +/- 2 and 43 +/- 5 ovulated ova/rat were found in animals primed with 3 and 10 IU FSH-CTP1, respectively. Because twice daily injections of WT-FSH (2.5 IU/injection) also increased the ovulatory potential of the ovary, the enhanced effectiveness of FSH-CTP1 appears to be related to its increased circulating half-life.(ABSTRACT TRUNCATED AT 400 WORDS)

Synthetic peptides based upon a three-dimensional model for the receptor recognition site of follicle-stimulating hormone exhibit antagonistic or agonistic activity at low concentrations.

Follicle-stimulating hormone (follitropin, FSH) belongs to a group of closely related glycoprotein hormones that contain two noncovalently linked dissimilar subunits designated alpha and beta. By using synthetic peptides, several receptor interaction sites in these hormones have been identified; however, the peptides have a reduced potency (lowest effective concentration of 10(-4) to 10(-5) M) relative to the hormone itself (10(-8) to 10(-11) M). This suggests that the peptides represent only a portion of a larger recognition site in the intact hormone that comprises parts of both the beta and the alpha chains. To develop peptides that exhibit FSH-antagonistic activity at low concentrations, we have constructed a three-dimensional model for FSH, which is based on an alignment of both the beta and the alpha chains of glycoprotein hormones with thioredoxin, for which x-ray diffraction data are available. This model resulted in the prediction of a conformational receptor-binding site in FSH, in which (parts of) three earlier proposed binding regions on the FSH molecule [namely, the regions FSH alpha-(34-37), with the amino acid sequence SRAY; FSH beta-(40-43), with the amino acid sequence TRDL; and FSH beta-(87-94), the "determinant loop" with the amino acid sequence CDSDSTDC] are located within 10 A of one another. On the basis of this model, peptides have been synthesized in which two of these binding regions are linked by a synthetic amino acid whose length was derived from the model, Ac-TDSDS-NH-(CH2)5-CO-SRAY-NH2 and Ac-SRAY-NH-(CH2)4-CO-TRDL-NH2. Both peptides inhibited FSH-induced cAMP production in Sertoli cells at 1000-fold lower concentrations (10(-7) M) than the peptides Ac-TRDL-NH2, Ac-SRAY-NH2, or Ac-TDSDS-NH2. In another peptide, Ac-TDSDS-NH-(CH2)5-CO-SRAY-NH-(CH2)4-CO-TRDL-NH2, all three binding regions have been linked. This peptide appeared to be a strong agonist of FSH action, as measured by the ability to stimulate cAMP production, at concentrations as low as 10(-7) M. The observation that a synthetic peptide, in which (parts of) three earlier described receptor interaction sites are combined according to the three-dimensional model, can mimic the action of FSH, at 10(-7) M, shows that this model is useful to predict a conformational receptor-binding site in FSH and that combination of only a few amino acid residues from the alpha and beta chains of FSH in a small synthetic peptide is sufficient to transduce a signal upon binding to the receptor.

Serine analogues of hFSH-beta-(33-53) and hFSH-beta-(81-95) inhibit hFSH binding to receptor.

Synthetic peptides corresponding to hFSH-beta-(33-53) and hFSH-beta-(81-95) each contain free sulfhydryl groups, inhibit binding of FSH to receptor and are partial agonists of estradiol synthesis in Sertoli cells. Recently, we have reported that sulfhydryl groups are important in FSH- receptor interaction and that peptides containing free sulfhydryl groups or disulfide bridges, such as glutathione, may nonspecifically inhibit FSH binding to receptor. In the present study, Cys residues of hFSH-beta-(33-53) and hFSH-beta-(81-95) were replaced by Ser residues and the peptides tested for their ability to inhibit binding of FSH to receptor. Results similar to those obtained previously with natural sequence peptides were obtained with the Ser analogs, indicating that Cys residues were not essential for binding inhibition. However, the partial agonist activity of the hFSH-beta-(33-53) and (81-95) in cultured Sertoli cells could not be detected when Cys residues were replaced by Ser. Thus, replacement of Cys residues with Ser does not effect receptor binding activity but is deleterious to the agonist activity of these peptides.

Deglycosylated human follitropin: characterization and effects on adenosine cyclic 3',5'-phosphate production in porcine granulosa cells.

Chemical deglycosylation of gonadotropins with hydrogen fluoride (HF) has facilitated the investigation of the structure-function relationship of the individual peptide and oligosaccharide moieties in the mechanism of hormone action. These studies have dealt almost exclusively with lutropin or human choriogonadotropin. We report here the chemical characterization and biological properties of deglycosylated human follitropin (degly hFSH). Results indicate that deglycosylation of hFSH by HF removes 89% of the total carbohydrate without disruption of the peptide chain or significant loss of amino acid residues. However, a change in the conformation of the molecule was observed by measurement of the far-ultraviolet circular dichroic spectrum. The degly hFSH showed a 44% reduction in binding when tested in a FSH radioimmunoassay utilizing a polyclonal antibody. Binding of the degly hFSH to FSH-responsive tissues showed that the altered hormone bound with equal or better avidity than the intact hormone while the association constants were approximately the same for both preparations. The degly hFSH alone did not stimulate the FSH-stimulatable adenylyl cyclase (AC) activity of cellular homogenates of small follicle porcine granulosa cells. Furthermore, degly hFSH was a potent antagonist of hFSH-stimulatable AC activity when coincubated with intact hFSH. In intact granulosa cells, both the hFSH and the degly hFSH stimulated cAMP production and release by these cells. However, the degly hFSH was one-tenth as effective as the intact hormone.(ABSTRACT TRUNCATED AT 250 WORDS)

Photoaffinity labeling of the follitropin receptor.

A photoactivatable derivative of human follitropin was used to identify the follitropin receptor on porcine granulosa cells. The hormone was condensed with a heterobifunctional reagent, the N-hydroxysuccinimide ester of 4-azidobenzoylglycine, and radioiodinated. The 125I-labeled hormone (125I-hormone) derivative associated with the same number of receptors as 125I-hormone itself, but with a slightly lower Ka, 1.12 X 10(10) M-1 compared with 1.4 X 10(10) M-1 for the 125I-hormone. The binding could be blocked with untreated hormone. Its alpha and beta subunits could be cross-linked to produce alpha beta dimer by photolysis. When the 125I-hormone derivative bound to the cells was photolyzed for crosslinking and the products resolved by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels under reducing conditions, two new bands (106 and 61 kDa) of lower electrophoretic mobility appeared in addition to the alpha, beta, and alpha beta bands. Formation of these crosslinked complexes required photolysis, and the 125I-hormone derivative specifically bound to cells bearing the receptor. Binding could be blocked by excess untreated follitropin but not with human choriogonadotropin and thyrotropin. Under nonreducing conditions, one major band (104 kDa) of cross-linked complexes appeared. Upon reduction with dithiothreitol and second-dimensional electrophoresis, the 104-kDa band produced two smaller complexes of 75 and 61 kDa, indicating the loss of two components and the existence of intercomponent disulfides. Successful production of the 104-kDa complex requires blocking of free sulfhydryl groups with N-ethylmaleimide. It is, however, independent of various protease inhibitors or the temperature and the time period of hormone incubation with cells or the plasma membrane fraction. The mass estimates and the interaction with the hormone of the photoaffinity-labeled components are discussed.

A specific FSH receptor in rat granulosa cells: properties of binding in vitro.

The specific binding of [125I]iodoFSH to granulosa cells collected from immature, hypophysectomized, DES-treated rats, was studied in vitro. Specific binding occurred after 5 min and reached maximum after 3 h of incubation at 37 C. Non specific binding was very low (less than 10% of the total binding). The [25I]iodoFSH remained tightly associated with the receptor at pH 7.5, but was rapidly dissociated at pH 5. Unlabeled hFSH competitively inhibited [125I]iodoFSH binding. Kinetic analyses of equilibrium binding experiments gave an apparent association constant (Ka) of 1.34 (+/- 0.31) x 10(10)M-1 [mean (+/- SE)] and a number of binding sites per cell of (NB) 1, 130 +/- 70 (mean +/- SE). Rat prolactin, wheat germ agglutinin, and concanavalin-A did not compete with [125I]iodoFSH, but hLH, hCG, and rTSH competed at doses 300- to 900-fold higher than those of hFSH. Granulosa cells isolated from adult DES-treated rats, as well as cells collected from medium and preovulatory follicles of proestrous rats, gave Ka and NB values similar to those described above. A comparative study of rabbit granulosa cells indicated a much lower binding affinity compared with those from the rat.