Human pluripotent stem cell-derived cells endogenously expressing follicle-stimulating hormone receptors: modeling the function of an inactivating receptor mutation.

Follicle-stimulating hormone (FSH) is crucial in the development and regulation of reproductive functions. The actions of human FSH and its receptor (FSHR) and mutations therein have mainly been studied using in vivo models, primary cells, cancer cells and cell lines ectopically expressing the FSHR. To allow studies of endogenous FSHR function in vitro, we differentiated FSHR-expressing cells from human pluripotent stem cells. FSH stimulation of the wild-type (WT), but not the inactivating Finnish founder mutant (A189V) receptor, activated the canonical cyclic adenosine monophosphate (cAMP)-dependent signaling pathway and downstream mediators. To investigate protein-protein interaction partners of FSHR at resting state and upon FSH stimulation, we expressed FSHR in HEK293 cells followed by affinity purification mass spectrometry analyses. We found 19 specific high-confidence interacting proteins for WT FSHR and 14 for A189V FSHR, several of which have been linked to infertility. Interestingly, while only WT FSHR interacted with FSH, insulin-like growth factor 1 receptor (IGF1R), for example, interacted with both WT and A189V FSHR upon FSH stimulation. In conclusion, our protocol allows detailed studies of FSH action and disease modeling in human cells endogenously expressing FSHR.

Does long in vitro culture promote large for gestational age babies?

STUDY QUESTION: Does the length of time during which embryos are cultured in vitro affect the birthweight of the infants? SUMMARY ANSWER: The duration of the embryo culture period is a significant factor in determining the birthweight of the infants. WHAT IS ALREADY KNOWN: IVF children show adverse perinatal outcome when compared with the general population and increased incidence of preterm birth and low birthweight is commonly observed. STUDY DESIGN, SIZE, DURATION: A retrospective cross-sectional cohort study including 1079 infants born after treatment at the Family Federation of Finland Fertility Clinic in Helsinki, between 2000 and 2010. PARTICIPANTS, SETTING, METHODS: All singleton IVF children were included. The gestation- and gender-adjusted birthweights of the babies were analyzed according to mother's age, BMI, and parity, type of treatment (IVF or ICSI), main cause of infertility and embryo culture period. Two outcomes were investigated: the birthweight and the proportion of small and large for gestational age (SGA and LGA) infants. Multiple linear regression analysis was performed to show the significance of each individual factor on determining the birthweight of the babies born. MAIN RESULTS AND THE ROLE OF CHANCE: In the study group as a whole, the distribution of the SGA and LGA babies showed no deviation from the growth charts of the general population. However, when the birthweight of the children was analyzed according to the length of embryo culture from Day 2 to Days 5-6, an increase in the proportion of LGA babies was found (D2 9.4%, D3 11.5%, D5-6 18.8%; D2 n = 871, D3 n = 139, D5-6 n = 69). Multiple linear regression analysis showed that BMI (P < 0.001) and parity (P < 0.001) of the mother, as well as the embryo culture period (P = 0.007) had a significant effect on the birthweight. The value of the adjusted R(2) was 0.437. LIMITATIONS, REASONS FOR CAUTION: Small number of D5-6 infants and a lack of pregnancy-associated factors contributing to birthweight. WIDER IMPLICATIONS OF THE FINDING: This study warrants larger studies to analyze the birthweight of the IVF children, particularly after blastocyst culture. STUDY FUNDING/COMPETING INTEREST: The study was funded by the Family Federation of Finland, Fertility Clinic Helsinki. No competing interests.

Association between sequence variations in genes encoding human zona pellucida glycoproteins and fertilization failure in IVF.

BACKGROUND: The zona pellucida (ZP) has multiple roles in reproductive processes, including oocyte maturation, fertilization and implantation. We used, for the first time, a genetic approach to study whether human ZP genes possess structural alterations in women with unsuccessful IVF trials. In theory, this may result in gradual reduction of sperm-zona interaction and eventually in total fertilization failure (TFF). METHODS: Eighteen infertile women (TFFs) whose IVF did not result in any fertilized oocytes, whereas fertilization by ICSI was successful, were screened for mutations in ZP genes by means of conformation-sensitive gel electrophoresis. Twenty-three fertilizers in IVF (FIVFs) and 68 women with proven fertility (WPFs) constituted the two control groups. RESULTS: Altogether, 20 sequence variations were found in the ZP genes. Two variations in ZP3, one in the regulatory region (c. 1-87 T --> G) and one in exon 6 [c. 894 G --> A (p. K298)] existed more frequently in TFFs than in FIVF and WPF groups (P-values 0.027 and 0.008, respectively). CONCLUSIONS: Our study on ZP genes of infertile women revealed a high degree of sequence variations. This may reflect gradual reduction of fertility among TFFs, but the putative roles and influences of single variations can only be hypothesized.

Cell death and its suppression in human ovarian tissue culture.

In women with premature ovarian failure, fertility may be preserved by ovarian tissue culture in vitro. However, techniques for tissue culture and follicle maturation have remained suboptimal. Our aim was to characterize ovarian tissue degeneration in cultures and to establish a model for cell death research in cultured ovarian tissue. Precise knowledge on the process resulting in cell death in cultured ovarian tissue will ultimately facilitate work aimed at improving long-term culture conditions. Ovarian tissue apoptosis was studied in a serum-free culture model in which nuclear DNA fragmentation was shown to occur within 24 h of the start of the culture. Activation of caspase-3 was detected in some stromal cells and a few oocytes. Since not all of the tissue exhibited signs of apoptosis and since DNA fragmentation increased over time, the tissue probably gradually dies by apoptosis. The antioxidant N-acetyl-L-cysteine (NAC; 25, 50 and 100 mmol/l) was found to inhibit this apoptosis. Thus, apoptosis appears to play a critical role in the degeneration of human ovarian cortical tissue cultures, and this cell death can be suppressed by NAC. The present tissue culture model can be used for identifying components capable of inhibiting cell death in vitro.

The predictive value of pronuclear morphology of zygotes in the assessment of human embryo quality.

BACKGROUND: Recent studies have shown that zygote morphology could be used for the assessment of human embryo quality. Pronuclear (PN) morphology is based on certain distinct features seen in zygotes 16-18 h after fertilization. In the present study PN stage morphology was assessed and combined with a single embryo transfer in order to investigate whether currently used zygote classifications are able to predict embryo quality and implantation rates. METHODS AND RESULTS: Zygotes were analysed according to two different classification systems. In the first, a total of 764 zygotes was analysed according to the degree of polarization of nucleolar precursor bodies (NPB). Zygotes with unpolarized PN (i.e. scattered localization of NPB) showed significantly slower (P < 0.005) cleavage rates (38.9%) than zygotes having at least one pronucleus polarized (57.3% and 54%). However, there was no difference in the pregnancy rate in 105 single embryo transfers between the groups. The appearance of a cytoplasmic halo was related to embryo morphology. Embryos derived from halo-positive zygotes had significantly better (P < 0.05) morphology (60.9%) compared to halo-negative derived embryos (52.2%), but in terms of pregnancy rates no difference was found. A total of 1520 zygotes was analysed according to a second classification system, which was based on the number and distribution of NPB. In the comparative analysis, none of the six different classes produced superior quality embryos or higher pregnancy rates in 144 single embryo transfers. CONCLUSIONS: Our results indicate that there are no significant differences in embryo quality or implantation/pregnancy rates between proposed zygote classes.

The effects of insulin, and insulin-like growth factors I and II on human ovarian follicles in long-term culture.

The aim of this study was to investigate the effects of insulin and insulin-like growth factors I and II (IGF-I and IGF-II) on human ovarian follicles in vitro. Ovarian cortical tissue slices (0.1-0.3 cm) were cultured for 7 or 14 days on an artificial extracellular matrix and with FSH. The ovarian tissue cultures were stimulated by insulin (33 ng/ml), IGF-I (20 or 50 ng/ml) or IGF-II (20 ng/ml). Combined effects of IGF-I (20 ng/ml) or IGF-II (20 ng/ml) and insulin (33 ng/ml) were also studied. Proliferating cell nuclear antigen (PCNA) was selected for immunohistochemical examination activation of the mitotic cell cycle in granulosa cells. After 1 week of culture the number of follicles had decreased in all cases. After 2 weeks of culture the number of healthy follicles had decreased dramatically in control cultures. However, the loss of follicles could be prevented with insulin and IGFs. The number of atretic follicles was significantly lower in insulin cultures compared with control cultures after 2 weeks. The proportion of primary follicles was significantly increased in cultures treated with insulin, IGF-I (50 ng/ml) or IGF-II (20 ng/ml) compared with control cultures after 2 weeks. A similar effect was seen after co-treatment with IGF-II and insulin. There were significantly more PCNA-positive follicles in IGF-I cultures than in control cultures. These results suggest that insulin, IGF-I and IGF-II may act as survival factors for early stage human follicles. IGFs may also be involved in activation of the mitotic cell cycle of granulosa cells.

Luteal phase start of low-dose FSH priming of follicles results in an efficient recovery, maturation and fertilization of immature human oocytes.

In this prospective study we investigated whether the maturation and fertilization of immature oocytes can be improved by administration of recombinant follicle stimulating hormone (rFSH) starting in the late luteal phase in two groups of women: group 1 (n = 6) women with regular menstrual cycles; and group 2 (n = 6) women with irregular cycles and polycystic ovaries (PCO) on ultrasound examination. Low-dose (37.5 IU) rFSH was commenced 11 days after LH surge during a spontaneous menstrual cycle and on the ninth day of progesterone administration in an irregular cycle. Recombinant FSH was continued until the leading follicle was approximately 10 mm in diameter. The oocytes were retrieved after withdrawing rFSH for 2-5 days. In total, 136 oocytes were recovered (group 1, 67 oocytes; group 2, 69 oocytes). Nine of the oocytes from PCO women were atretic at retrieval. Oocytes complete with cumulus cells were cultured for 44 h in complex tissue culture medium supplemented with gonadotrophins and fetal calf serum. After maturation, the cumulus cells were removed and metaphase II oocytes were injected with spermatozoa. Respectively, the oocyte maturation and fertilization rates were 64 and 72% in group 1, and 78 and 57% in group 2 (not significant). After fertilization, the zygotes (group 1, n = 22; group 2, n = 11) and cleavage stage embryos (group 1, n = 9; group 2, n = 15) were frozen in propanediol. All women except one (11/12) had approximately five zygotes or cleaved embryos frozen. The viability of in-vitro matured frozen-thawed embryos was generally poorer than that (81%) seen after conventional intracytoplasmic sperm injection, with 61% survival in group 1 and 23% in group 2. Fifteen embryo transfers resulted in one miscarriage at 6 weeks gestation. The late luteal start of low-dose rFSH yielded a good number of immature oocytes in women with both regular and irregular cycles. Two out of three of these oocytes matured and fertilized. However, cryosurvival of the zygotes and cleaved embryos was unsatisfactory and thus cryopreservation of in-vitro matured embryos may not be an optimal procedure.

Comparison of two recombinant follicle-stimulating hormone preparations in in-vitro fertilization: a randomized clinical study.

A randomized comparison of two recombinant human follicle-stimulating hormone (recFSH) preparations (Gonal-F and Puregon) in ovarian stimulation for in-vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) was carried out at the Infertility Clinic of the Family Federation of Finland. A total of 348 women (aged 22-43 years) suffering from infertility due to miscellaneous causes was recruited. Of these, 344 underwent stimulation using equal starting doses (150 IU/day: Gonal-F n = 164, Puregon n = 158 or 300 IU/day: Gonal-F n = 8, Puregon n = 14) after down-regulation with intranasal buserelin from the mid-luteal phase. Similar clinical pregnancy rates were achieved with both preparations; 33.5% per cycle and 37.4% per embryo transfer (24.5% one-embryo and 75.5% two-embryo transfers, n = 147) with Gonal-F (150 IU/day) and 32.9% per cycle and 36.4% per embryo transfer (30.1% one-embryo and 69.9% two-embryo transfers, n = 145) with Puregon (150 IU/day). The ongoing cumulative pregnancy rates after frozen-thawed embryo transfer were 35.4% with Gonal-F and 37.7% with Puregon. Six cycles were cancelled because of a low response (three in each group). Similar numbers of oocytes were obtained in both groups; 13.0 with 150 IU/day and 6.1 with 300 IU/day Gonal-F, and 12.4 with 150 IU/day and 7.1 with 300 IU/day Puregon. The fertilization and cleavage rates and the incidence of moderate or severe ovarian hyperstimulation syndrome (Gonal-F, 2.0% and Puregon, 0.7%) were also similar. Gonal-F and Puregon were equally and highly effective in stimulation for IVF and ICSI.

Human growth differentiation factor 9 (GDF-9) and its novel homolog GDF-9B are expressed in oocytes during early folliculogenesis.

Growth differentiation factor 9 (GDF-9) is a transforming growth factor-beta family member that is required for normal folliculogenesis in female mice, but its role as a regulator of human fertility is still unclear. We determined here by in situ hybridization and immunohistochemical analyses the localization of the GDF-9 messenger ribonucleic acid (mRNA) and protein during human folliculogenesis. The GDF-9 transcripts were not detected in primordial follicles, but they are abundantly expressed in primary follicles in frozen sections of ovarian cortical tissue material obtained at laparoscopic surgery. We raised antipeptide antibodies against GDF-9 and showed by immunohistochemical studies on paraffin sections of whole human ovaries that the GDF-9 protein is most abundantly expressed in primary follicles. We recently demonstrated that a novel GDF-9-related factor, GDF-9B, is coexpressed with GDF-9 during murine folliculogenesis. We now isolated human GDF-9B complementary DNA and genomic clones and report the unusually restricted expression pattern of human GDF-9B. The human GDF-9B transcript can be detected only in the gonads by RT-PCR analysis, and in situ hybridization studies indicate that it is not expressed in small primary follicles but, rather, in the oocytes of late primary follicles. Functional studies using the Xenopus laeuis embryo model indicate that unlike the transforming growth factor-beta family members activin and bone morphogenetic protein-4, neither GDF-9 nor GDF-9B affects mesoderm induction, suggesting that they may use signaling pathways distinct from those well defined for activin and bone morphogenetic protein-4. We conclude that 1) both GDF-9 mRNA and protein are abundantly expressed in oocytes of primary follicles in human ovary, suggesting that the GDF-9 transcript is translated at this early stage of folliculogenesis; 2) human GDF-9B is specifically expressed in gonads at low levels; and 3) the expression of GDF-9 mRNA begins slightly earlier than that of GDF-9B in the human oocytes during follicular development. Our results are consistent with the suggestion that GDF-9 and GDF-9B may regulate human folliculogenesis in a manner specific to the ovary.

Co-ordinate expression of activin A and its type I receptor mRNAs during phorbol ester-induced differentiation of human K562 erythroleukemia cells.

Activins were originally isolated based on their ability to stimulate follicle-stimulating hormone secretion but later they have been shown to regulate a number of different cellular functions such as nerve cell survival, mesoderm induction during early embryogenesis as well as hematopoiesis. We studied the regulation of activin A, a homodimer of betaA-subunits, mRNA and protein in K562 erythroleukemia cells, which are known to be induced toward the erythroid lineage in response to activin or TGF-beta or toward the megakaryocytic lineage by the phorbol ester protein kinase C activator 12-O-tetradecanoylphorbol-13-acetate (TPA). Here we show by Northern blot analysis as well as by Western and ligand blotting that TPA strongly promotes activin betaA-subunit mRNA and activin A protein expression in K562 cells in time- and concentration dependent manner. In contrast, neither activin A nor TGF-beta induced betaA-subunit mRNA expression during erythroid differentiation in K562 cells. Interestingly, whereas activin type II receptors are not regulated during K562 cell differentiation (Hilden et al. (1994) Blood 83, 2163-2170), we now show that the activin type I and IB receptor mRNAs are clearly induced by TPA but not by activin or TGF-beta. We also show that the inducing effect of TPA on expression of activin betaA-subunit mRNA is potentiated by the protein kinase A activator 8-bromo-cAMP. We conclude that activin A and its type I receptors appear to be co-ordinately up-regulated during megakaryocytic differentiation of K562 cells.

Testicular biopty gun needle biopsy in collecting spermatozoa for intracytoplasmic injection, cryopreservation and histology.

Using testicular spermatozoa from either open biopsy (29 cycles) or biopty gun needle biopsy (49 cycles), a total of 81 intracytoplasmic sperm injection (ICSI) cycles among 57 couples were carried out from January, 1994 to September, 1997. In six cycles, no spermatozoa were obtained, and in three cycles spermatozoa from both needle and open biopsies were used. The fertilization (37% after open and 41% after needle biopsy) and pregnancy rates (29% per embryo transfer compared with 16% per embryo transfer) were similar after both open and needle biopsies. Five pregnancies were achieved among the 14 couples with non-obstructive azoospermia of the male partner, four of these after needle biopsy. It was possible to use cryopreserved testicular spermatozoa after both needle and open biopsies, and one pregnancy started after using cryopreserved testicular spermatozoa in both groups. Histological needle biopsy was carried out in 62 cases, and they were all diagnostic, giving 15-20 cross-sections of seminiferous tubuli per biopsy. Testicular needle biopsy using a 14 gauge biopsy needle gave a sufficient amount of tissue and spermatozoa for ICSI, cryopreservation and histology, even in non-obstructive azoospermia. This technique is simpler and cheaper than open biopsy and, hence, it can be regarded as the optimal method for the retrieval of testicular spermatozoa.

Hormonal and photoperiodic modulation of testicular mRNAs coding for inhibin/activin subunits and follistatin in Clethrionomys glareolus, Schreber.

Photoperiodic and hormonal modulation of mRNAs for testicular inhibin/activin subunits and follistatin were studied in a seasonally breeding rodent, the bank vole (Clethrionomys glareolus). Photoperiod-induced testicular regression had no effect on the relatively low steady-state levels of follistatin mRNA. Inhibin alpha (I alpha) and beta B (I beta B) mRNA levels were significantly higher in regressed than in active gonads, but inhibin beta A was undetectable. The effect of gonadotropin administration on testicular weight and mRNA concentrations differed between the sexually active and quiescent voles. Neither FSH (1.2 U/kg; s.c. for 5 days) nor hCG (600 IU/kg; s.c. for 5 days) affected testicular weight in sexually active voles, whereas both gonadotropins significantly increased testicular weight in photo-regressed individuals. FSH had no effect on I alpha or I beta B mRNA concentrations in the active testes, whereas excessive hCG challenge induced a decrease in the steady-state levels of these mRNAs. FSH induced an increase in I alpha mRNA concentrations in the regressed gonad, whereas both gonadotropins concomitantly down-regulated I beta B mRNA levels. In conclusion, the high expression of I alpha and I beta B mRNA in the regressed testis imply autocrine and paracrine roles for inhibin/activin in the quiescent gonad of seasonal breeders. Inhibin alpha-subunit expression is at least partly under the control of FSH in the bank vole testis.

Activin and bone morphogenetic protein (BMP) signalling during tooth development.

Activins and bone morphogenetic proteins (BMPs) belong to the transforming growth factor-beta (TGF-beta) superfamily of structurally related cytokines. Results of recent in vivo and in vitro studies suggest that activin A, several BMPs, and their cell surface receptors may participate in tooth development. In addition, follistatin an extracellular protein that may interact with activin and BMP signaling, appears to be involved. This review focuses on recent advances in relation to the roles of activin/BMP receptors, their ligands and extracellular modifiers during tooth development.

The activin-binding protein follistatin is expressed in developing murine molar and induces odontoblast-like cell differentiation in vitro.

It has recently been shown that mice deficient in activin-beta A subunits and follistatin exhibit major defects in dentition. To increase understanding of the roles played by these molecules during tooth development, we determined the temporospatial expression of activin-beta A subunit and follistatin messenger RNA and their corresponding proteins in developing murine molars (between day E 14 and 2 days after birth). The effects of recombinant human activin A and its binding protein follistatin on odontoblast differentiation were also studied in cultures of dental papillae (DP) isolated from the mandibular first molars of E-17-day mice. In situ hybridization indicated that transcripts for activin-beta A subunit were abundant in pre-odontoblasts at the tips of forming cusps prior to odontoblast terminal differentiation, and transcripts for follistatin in overlying inner enamel epithelial cells (pre-ameloblasts). Pre-odontoblasts were also weakly immunoreactive in relation to activin-beta A subunit, pre-ameloblasts in relation to follistatin. When follistatin was added at different concentrations to a DP culture model (2-14 nmol/DP) together with heparin at constant concentration, differentiation of odontoblast-like cells was induced, as evidenced by polarization and deposition of extracellular matrix in vitro, to extents depending on the follistatin concentration. In contrast, the addition of activin A (2 nmol/DP) had no effect on the differentiation parameters studied. These findings suggest that the activin-follistatin system regulates odontoblast differentiation during tooth development. In particular, we suggest that binding of endogenous activin A by follistatin may allow odontoblast terminal differentiation to occur.

Identification of ovarian granulosa cells as a novel site of expression for bone morphogenetic protein-3 (BMP-3/osteogenin) and regulation of BMP-3 messenger ribonucleic acids by chorionic gonadotropin in cultured human granulosa-luteal cells.

Bone morphogenetic proteins (BMP) belong structurally to the transforming growth factor-beta superfamily comprising several growth and differentiation factors such as inhibin, activin, and Müllerian inhibitory factor that regulate ovarian function. We studied here the potential expression of BMP-2, -3, and -4 messenger RNAs (mRNAs) in isolated human granulosa cells obtained at oocyte retrieval for in vitro fertilization. Freshly isolated granulosa cells were found to express BMP-3 (also known as osteogenin) mRNAs but not those of BMP-2 or -4. The BMP-3 transcripts were detected with RT-PCR amplification followed by Southern blot hybridization, as well as by Northern and dot blot hybridization analyses. To investigate whether BMP-3 mRNAs are hormonally regulated, cultures of human granulosa-luteal (GL) cells were treated with different concentrations of purified human chorionic gonadotropin (hCG) at varying stages of culture. hCG decreased BMP-3 mRNA levels from the first day of the culture up to day 5. Time-dependence studies showed that a clear decrease in BMP-3 mRNA levels was evident at 24 h after hCG treatment, and that the effect of hCG was concentration dependent with 3 ng/mL hCG decreasing significantly (P < 0.05) BMP-3 mRNA levels. Furthermore, the cAMP analog, 8-bromo-cAMP (8-Br-cAMP), which activates protein kinase-A, and 12-0-tetradecanoylphorbol 13-acetate, an activator of protein kinase-C, both markedly decreased BMP-3 mRNA levels in an 8-h treatment. We conclude that: 1) BMP-3 mRNAs are expressed in human granulosa cells; 2) their steady state levels are hormonally regulated in cultured human GL cells as evidenced by the ability of hCG to markedly decrease BMP-3 transcript levels; and (3) that activation of both protein kinase-A-and protein kinase-C-mediated signaling pathways also results in a decrease in BMP-3 mRNA levels in GL cells. We suggest that BMP-3, like several other members of the transforming growth factor-beta superfamily, is a potential local regulator of female gonadal function.

Differential regulation of inhibin/activin alpha- and beta A-subunit and follistin mRNAs by cyclic AMP and phorbol ester in cultured human granulosa-luteal cells.

Granulosa cell-derived inhibin A (a dimer of alpha- and beta A-subunits), activin A (a homodimer of beta A-subunits) and the activin-binding protein follistatin are important regulators of human ovarian steroidogenesis. We here studied how 8-bromo-cAMP (8br-cAMP), a protein kinase A activator, and 12-O-tetradecanoylphorbol 13-acetate (TPA), a protein kinase C activator, affect the steady-state levels of alpha- and beta A-subunit and follistatin mRNAs in cultured human granulosa-luteal cells. 8br-cAMP induced alpha- and beta A-subunit and follistatin steady-state mRNA levels in a time- and concentration-dependent manner. The levels of alpha-subunit mRNAs were stimulated by 8br-cAMP in a sustained manner with a maximal induction seen at the time points 24 and 48 h. By contrast, beta A-subunit and follistatin mRNA levels were rapidly and transiently induced by 8br-cAMP with maximal effects observed at 3 h and 8 h, respectively. TPA did not affect basal alpha-subunit mRNA levels but it rapidly induced beta A-subunit mRNAs at 3 h and the stimulation was still evident at 48 h. TPA induced follistatin mRNA levels with kinetics similar to 8br-cAMP but to a lesser extent. Moreover, 8br-cAMP and TPA stimulated beta A-subunit and follistatin mRNA levels synergistically at 3 h. By contrast, TPA had a potent inhibitory effect on 8br-cAMP- and hCG-induced alpha-subunit levels. Neither 8br-cAMP nor TPA regulated inhibin/activin beta B-subunit mRNA levels. Taken together the activation of protein kinase-A and -C by 8br-cAMP and TPA, respectively, lead to clearly differential responses in the steady-state levels of inhibin activin alpha- and beta A-subunit and follistatin mRNAs. These results suggest that the inhibin A vs. activin A ratio as well as follistatin levels are regulated by multiple second-messenger pathways in the human ovary.

Regulation of the activin-binding protein follistatin cultured human luteinizing granulosa cells: characterization of the effects of follicle stimulating hormone, prostaglandin E2, and different growth factors.

Regulation of the activin-binding protein follistatin (FS) by recombinant human (rh) FSH, prostaglandin E2 (PGE2), and several polypeptide growth factors was examined in cultures of human granulosa-luteal (GL) cells obtained from in-vitro fertilization patients at oocyte retrieval. Northern and dot blot hybridization analyses demonstrated that both rhFSH and PGE2 caused stimulatory effects on FS mRNA levels in a culture stage-, time-, and concentration-dependent manner. An 8-h stimulation with rhFSH (100 ng/ml) significantly increased FS mRNA levels on Days 5 and 7 of culture and PGE2 (10(-6)M) on Days 2, 4, and 7. The stimulatory effect of rhFSH and PGE2 on FS mRNA levels were rapid and transient. Maximal inductions occurred 8 h after stimulation, whereas weak or no stimulatory effects were seen at 24 or 48 h. PGF2 alpha did not affect FS mRNA levels at any time point studied. Treatment of the cells with the protein synthesis inhibitor cycloheximide prior to rhFSH stimulation did not inhibit the rapid induction of FS mRNAs, but it prevented the decline at 24 h. Both rhFSH and PGE2 clearly also increased the levels of secreted FS proteins are detected by immunoprecipitation studies with a specific antibody. The effects of the polypeptide growth factors epidermal growth factor (EGF); transforming growth factor beta 1 (TGF beta 1), and activin A on FS mRNA levels were also examined. TGF beta 1 and activin A had no effect on basal FS expression at any concentration or time point studied. An 8-h stimulation with EGF increased FS mRNA levels, but the effect was weaker than those caused by rhFSH and PGE2. We conclude that rhFSH and PGE2 induce FS mRNA and protein in human cultured GL cells. EGF is able to induce FS mRNA to a lesser extent than are rhFSH and PGE2, whereas PGF2 alpha, TGF beta 1, and activin A do not affect basal FS mRNA levels in human cultured GL cells. This study together with our previous report on the stimulatory effect of hCG on FS levels suggest that in the luteal phase of the human menstrual cycle, FS expression in granulosa cells is likely to be positively controlled by luteotropic factors such as gonadotropins and PGE2. Consequently, elevated FS levels may support the survival of the human CL since FS is known to prevent the antisteroidogenic effects of activin in human GL cells.

Regulation of inhibin/activin subunit messenger ribonucleic acids (mRNAs) by activin A and expression of activin receptor mRNAs in cultured human granulosa-luteal cells.

Recent studies have indicated that activin and inhibin may act as local regulators of cell growth and steroidogenesis in the human ovary. We studied the effect of recombinant human activin A and purified bovine inhibin A on the steady state messenger RNA (mRNA) levels of the inhibin/activin alpha-, beta A-, and beta B-subunits in cultured granulosa-luteal (GL) cells from preovulatory ovarian follicles of women undergoing in vitro fertilization. Activin A induced the expression of a 4.8-kilobase beta B-subunit mRNA transcript without affecting basal expression levels of the alpha- and beta A-subunit mRNAs. It stimulated beta B-subunit mRNA levels in a concentration- and time-dependent manner. Maximal stimulation of beta B-subunit mRNA levels was obtained with 30-100 ng/ml activin A. The level of beta B-subunit mRNAs increased significantly 8 h after stimulation, rising gradually thereafter to a maximum at 48 h. Inhibin A did not affect the mRNA levels of any inhibin/activin subunits, nor did it inhibit the effect of activin A. Recombinant human follistatin did not affect basal beta B-subunit mRNA levels, but it neutralized the effect of activin A. Although hCG induces inhibin/activin alpha- and beta A-subunit mRNA levels in human GL cells, it did not increase basal beta B-subunit levels. By contrast, it inhibited activin A-induced beta B-subunit mRNA levels. On the other hand, activin A decreased hCG-induced mRNA levels of the inhibin alpha-subunit and cytochrome P450 side-chain cleavage (P450scc) enzyme, an important rate-limiting enzyme in human GL cell progestin synthesis. Moreover, we observed by Northern blot analysis that cultured human GL cells as well as freshly isolated preovulatory granulosa cells express the specific mRNAs for all currently known serine/threonine kinase activin receptors, i.e. activin receptors I, IB, II, and IIB. Our results suggest that in GL cells, activin A may locally stimulate synthesis of the beta B-subunit in an autocrine or paracrine manner, and that in human ovary, regulation of the beta B-subunit differs from that of the alpha- and beta A-subunits.

Activin disrupts epithelial branching morphogenesis in developing glandular organs of the mouse.

We report that activin profoundly alters epithelial branching morphogenesis of embryonic mouse salivary gland, pancreas and kidney rudiments in culture, indicating that it may play a role as a morphogen during mammalian organogenesis. In developing pancreas and salivary gland rudiments, activin causes severe disruption of normal lobulation patterns of the epithelium whereas follistatin, an activin-binding protein, counteracts the effect of activin. In the kidney, activin delays branching of the ureter bud and reduces the number of secondary branches. TGF-beta induces a pattern of aberrant branching in the ureter bud derived epithelium distinct from that seen for activin. Reverse-transcriptase polymerase chain reaction, Northern hybridization and in situ hybridization analyses indicate that these developing tissues express the mRNA transcripts for activin subunits, follistatin or activin receptors. Our results are suggestive of a potential role for the activin-follistatin system as an intrinsic regulator of epithelial branching morphogenesis during mammalian organogenesis.

Regulation of inhibin alpha- and beta A-subunit messenger ribonucleic acid levels by chorionic gonadotropin and recombinant follicle-stimulating hormone in cultured human granulosa-luteal cells.

We studied the effects of recombinant human FSH (rhFSH) and purified hCG on the steady state messenger ribonucleic acid (mRNA) levels of inhibin alpha- and beta A-subunits in cultured granulosa-luteal cells of preovulatory ovarian follicles obtained from women undergoing in vitro fertilization. Specific mRNA transcripts for the alpha- and beta A-subunits were detected in Northern and dot blot filter hybridization analyses, and the levels of these mRNAs were induced by rhFSH and hCG in a distinct concentration- and time-dependent manner. The basal and hCG-stimulated alpha-subunit mRNA levels were first determined at 2- to 3-day intervals over a 3- to 10-day culture period after the initiation of the cultures. Both the basal and hCG-stimulated alpha-subunit mRNA levels declined steadily during culture, but the maximal relative stimulatory effect of hCG was observed on day 7 of culture. All subsequent experiments, therefore, were performed on days 6-8 of culture. Both gonadotropins induced alpha-subunit mRNA levels with slower kinetics than those of the beta A-subunit. Varying between experiments, rhFSH and hCG increased the expression of the alpha-subunit with a maximal effect of 2.5- to 5.7-fold and 1.7- to 7.2-fold, respectively, above basal levels 24-48 h after stimulation. rhFSH and hCG induced beta A-subunit mRNA levels with 3.0- to 5.8-fold and 2.3- to 8.6-fold increases above basal levels, respectively, at 2 h; thereafter, only moderate or no stimulation of the beta A-subunit mRNA levels could be detected at 7-48 h. Treatment of the cells with the RNA synthesis inhibitor actinomycin-D prevented the induction of alpha-subunit mRNA levels by hCG, and no significant differences were detected in the stability of alpha-subunit mRNA transcripts in hCG-treated cells vs. untreated cultures. This indicates that hCG induces transcription of the alpha-subunit gene rather than maintains the levels of preexisting transcripts. As the kinetics of induction of alpha- and beta A-subunit mRNAs by gonadotropins were different, we examined how the inhibition of protein synthesis affects the induction of alpha- and beta A-subunit mRNAs by hCG. Cycloheximide had no effect on basal alpha-subunit mRNA levels at 2 or 24 h. However, it inhibited at 24 h the induction of the alpha-subunit by hCG.(ABSTRACT TRUNCATED AT 400 WORDS)