hCG Treatment Research Articles

The relationship between Evi-1 expression and mouse ovarian follicular development

The ecotropic viral integration site-1 (Evi-1) is a transcription factor with two sets of zinc finger domains. It is an important regulator of the transforming growth factor beta superfamily. In the present study, we investigated the spatiotemporal expression patterns of Evi-1 using immunohistochemistry in ovaries from neonatal mice, gonadotropin-induced immature mice and mice in estrous cycle. Positive staining for Evi-1 was detected in the cytoplasm of oocytes. In postnatal mice, a high level of Evi-1 immunostaining was found from day 1 to 6, an intermediate level from day 10 to 16 and a low level on day 23. After gonadotropin treatment, Evi-1 was mainly expressed in small follicles and exhibited a very low level in large antral follicles. During the estrous cycle, the expression of Evi-1 was higher in diestrus and estrus than in proestrus and metestrus. Real-time PCR was performed to examine the relationship between Evi-1 mRNA and ovulation related genes (Ptgs2, Tnfaip6, Has2, Cd44, C1qbp). At 4h after hCG treatment, Evi-1 mRNA was down-regulated whereas ovulation related genes were up-regulated. Overall, the results indicate that Evi-1 is expressed in a stage-specific manner during ovarian follicular development and may be involved in early follicle development.

Administration of human chorionic gonadotropin at embryo transfer induced ovulation of a first wave dominant follicle, and increased progesterone and transfer pregnancy rates

We hypothesized that administration of hCG to recipients at embryo transfer (ET) would induce accessory CL, increase serum progesterone concentrations, and reduce early embryonic loss (as measured by increased transfer pregnancy rates). At three locations, purebred and crossbred Angus, Simmental, and Hereford recipients (n = 719) were assigned alternately to receive i.m. 1,000 IU hCG or 1 mL saline (control) at ET. Fresh or frozen-thawed embryos were transferred to recipients with a palpable CL on Days 5.5 to 8.5 (median = Day 7) of the cycle (Locations 1 and 2), or on Day 7 after timed ovulation (Location 3). Pregnancy diagnoses (transrectal ultrasonography) were done 28 to 39 d (median = 35 d) and reconfirmed 58 to 77 d (median = 67 d) post-estrus. At Location 1 (n = 108), ovaries were examined at pregnancy diagnosis to enumerate CL. More (P < 0.001) pregnant hCG-treated cows (69.0%) had multiple CL than pregnant controls (0%). Serum progesterone (ng/mL) determined at Locations 1 and 2 (n = 471) at both pregnancy diagnoses in pregnant cows was greater (P ≤ 0.05) after hCG treatment than in controls (first: 8.1 ± 0.9 vs 6.1 ± 0.8; second: 8.8 ± 0.9 vs 6.6 ± 0.7), respectively. Unadjusted pregnancy rates at the first diagnosis were 61.8 and 53.9% for hCG and controls. At the second diagnosis, pregnancy rates were 58.6 and 51.3%, respectively. Treatment (P = 0.026), embryo type (P = 0.016), and BCS (P = 0.074) affected transfer pregnancy rates. Based on odds ratios, greater pregnancy rates occurred in recipients receiving hCG, a fresh embryo (66.3 vs 55.5%), and having BCS >5 (62.3 vs 55.3%). We concluded that giving hCG at ET increased incidence of accessory CL, serum progesterone in pregnant recipients, and transfer pregnancy rates. Furthermore, we inferred that increased progesterone resulting from hCG-induced ovulation reduced early embryonic losses after transfer of embryos to recipients.

Effects of Induction of Ovulation with GnRH or hCG on Follicular and Luteal Blood Flow in Holstein–Friesian Heifers

The objective of this study was to compare the effects of gonadotropin-releasing hormone (GnRH) and human chorionic gonadotropin (hCG) on follicular blood flow (FBF) during the pre-ovulatory period and on luteal blood flow (LBF) during dioestrus in Holstein-Friesian heifers. Twelve animals were randomly divided into two groups and treated with either intramuscular injection of 100 μg GnRH or intravenous (IV) injection of 5000 IU hCG on Day 0 (oestrus, 48 h after administration of PGF(2α) ) to induce ovulation. Follicular size (FS), FBF and time of ovulation were recorded with colour Doppler sonography at 0, 1, 3, 6, 12 and 24 h after GnRH and hCG treatment. Luteal size (LS) and LBF were investigated on Day 9 and 12 after ovulation. Plasma samples were taken to determine total oestrogens (E(tot) ) and progesterone (P(4) ) after each examination. Ovulation occurred between 24 and 48 h after treatment in all animals. No difference (p > 0.05) was observed in FS between the two treatment groups. Follicular blood flow was higher in the hCG group than that was in GnRH group at 1 h after treatment (p < 0.01). Total oestrogens were also higher (p < 0.01) in the hCG group than GnRH group; however, this difference was only obvious at 12 h after treatment. No difference (p > 0.05) was observed in LS, LBF or P(4) levels on Day 9 and 12 between treatment groups. In conclusion, the results suggest that induction of ovulation with hCG and GnRH has a temporary effect on FBF and oestrogen levels while no effect on the size of corpora lutea, LBF and P(4) levels was observed.

Effect of GnRH agonist and hCG treatment on VEGF, angiopoietin-2, and VE-cadherin: trying to explain the link to ovarian hyperstimulation syndrome

This study evaluated the differentially modulated expression of vascular mediators in oocyte donors after hCG vs. GnRHa triggering, trying to understand ovarian hyperstimulation syndrome pathophysiology. Donors who received GnRH agonist triggering showed a statistically significant decrease in vascular endothelial growth factor in follicular fluid and in mRNA expression in granulosa cells, with no differences in angiopoietin 2 and vascular endothelial cadherin levels in serum or follicular fluid. This differential regulation of vascular endothelial growth factor by hCG might explain the higher likelihood of ovarian hyperstimulation syndrome following hCG administration compared with GnRH agonists.

Use of the natural cycle and vitrification thawed blastocyst transfer results in better in-vitro fertilization outcomes

To compare the IVF outcomes of vitrification-thawed blastocyst transfer cycles utilizing different endometrial preparation methods. We retrospectively assessed IVF outcomes in 611 patients (648 cycles) who underwent blastocyst frozen embryo transfer (FET) between January 2007 and December 2009. All embryos had been cryopreserved by a vitrification method following a previous IVF cycle. Patients were prepared for transfer by using either the natural cycle (n = 310/Group 1), the natural cycle with ovulation induction employing human chorionic gonadotropin (n = 134/Group 2), or a hormonally manipulated artificial cycle with estrogen and progesterone supplementation (n = 204/Group 3). Multivariate logistic regression analysis showed a significant difference in clinical pregnancy rate between Groups 3 (30.4%) and 1 (41.9%) (odds ratio [OR], 0.567; 95% confidence interval [CI], 0.379-0.847, P = 0.006) whereas the difference between Groups 2 and 1 was not significant (41.8% vs. 41.9%; OR, 0.683; 95% CI, 0.435-1.073; P = 0.098). Other significant variables affecting clinical pregnancy rate were the number of embryos transferred, the grade of transferred embryos, and maximal endometrial thickness. The results showed that, using vitrification-thawed blastocyst transfer, employment of natural cycles with or without hCG treatment was associated with better outcomes than was the use of hormonally manipulated cycles.

Epiregulin-dependent amphiregulin expression and ERBB2 signaling are involved in luteinizing hormone-induced paracrine signaling pathways in mouse ovary

Sustained EGF receptor (EGFR) phosphorylation by de novo synthesis of EGFR ligands plays an essential role in mediating luteinizing hormone (LH)-induced ovulation process in the preovulatory follicles (POFs). In the present study, the effect of epiregulin (EREG) on oocyte maturation and ovulation was investigated using Ereg knockout ( Ereg −/− ) mice congenic on a C57BL/6 background. Rate of spontaneous oocyte meiotic resumption of denuded oocytes (DOs) or cumulus cell-oocyte complexes (COCs) in vitro is similar between wild-type and Ereg −/− mice. However, gonadotropin-induced meiotic resumption in vivo is attenuated, and the number of COCs with expanded cumulus matrix and superovulated eggs dramatically decrease in Ereg −/− mice. Nonetheless, the number of eggs ovulated during normal estrus cycles and litter sizes in Ereg −/− mice are comparable to those of wild-type littermates. In contrast to other EGFR ligands, induction of amphiregulin ( Areg) mRNA is severely reduced in ovaries collected from Ereg −/− mice either after human chorionic gonadotropin (hCG) treatment in immature mice or LH surge in adults. Gonadotropin-induced EGFR and ERBB2 phosphorylation in ovaries is attenuated in immature Ereg −/− mice, and MAPK3/1 phosphorylation and prostaglandin synthase 2 (PTGS2) protein levels are reduced. This attenuation, however, is no longer detectable in adult Ereg −/− mice after LH surge. This study implicates that EREG mediates signals downstream of Areg mRNA expression and that EGFR-ERBB2 signals contributes to regulation of ovulation process.

Gonadotrophin-induced paracrine regulation of human oocyte maturation by BDNF and GDNF secreted by granulosa cells

In mammalian ovaries, diverse paracrine factors have been identified to mediate or modulate LH-induced changes during ovulation. Due to the difficulty in obtaining non-stimulated granulosa cells during IVF, little is known about the LH-induced paracrine factors in the human ovary. Based on earlier studies using murine ovarian cells showing the paracrine roles of brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) in promoting oocyte maturation, we investigated the expression of these ligands in human granulosa cells and their regulation of human oocyte development. Non-stimulated granulosa cells were obtained from non-stimulated IVM (in vitro maturation) patients after oocyte retrieval. Women undergoing non-stimulated IVM treatment at a mean age of 30.8 ± 1.3 (n = 10) were recruited for this study. Immature oocytes and granulosa cells were collected from IVF patients undergoing gonadotrophin stimulation and ICSI. Immunocytochemical analyses of granulosa cells were carried out to investigate expression profiles of BDNF and GDNF, together with real-time RT-PCR to analyze the gonadotrophin regulation of BDNF and GDNF transcript levels. In addition, immature oocytes were cultured to analyze the regulation of oocyte maturation by BDNF and GDNF. BDNF and GDNF were found to be expressed in non-stimulated granulosa cells. After gonadotrophin (FSH and/or hCG) treatment, transcripts levels for BDNF and GDNF were significantly increased (P < 0.05). In cultured immature oocytes, treatment with BDNF or GDNF promoted total yields of metaphase II oocytes. These findings demonstrate that FSH and hCG treatments augment the expression of BDNF and GDNF by granulosa cells and that these granulosa-cell-derived factors are candidate paracrine factors capable of promoting oocyte maturation.

A Role for Nuclear Factor Interleukin-3 (NFIL3), a Critical Transcriptional Repressor, in Down-Regulation of Periovulatory Gene Expression

The LH surge triggers dramatic transcriptional changes in genes associated with ovulation and luteinization. The present study investigated the spatiotemporal expression of nuclear factor IL-3 (NFIL3), a transcriptional regulator of the basic leucine zipper transcription factor superfamily, and its potential role in the ovary during the periovulatory period. Immature female rats were injected with pregnant mare's serum gonadotropin, treated with human chorionic gonadotropin (hCG), and ovaries or granulosa cells were collected at various times after hCG. Nfil3 mRNA was highly induced both in intact ovaries and granulosa cells after hCG treatment. In situ hybridization demonstrated that Nfil3 mRNA was highly induced in theca-interstitial cells at 4-8 h after hCG, localized to granulosa cells at 12 h, and decreased at 24 h. Overexpression of NFIL3 in granulosa cells inhibited the induction of prostaglandin-endoperoxide synthase 2 (Ptgs2), progesterone receptor (Pgr), epiregulin (Ereg), and amphiregulin (Areg) and down-regulated levels of prostaglandin E2. The inhibitory effect on Ptgs2 induction was reversed by NFIL3 small interfering RNA treatment. In theca-interstitial cells the expression of hydroxyprostaglandin dehydrogenase 15-(nicotinamide adenine dinucleotide) (Hpgd) was also inhibited by NFIL3 overexpression. Data from luciferase assays demonstrated that NFIL3 overexpression decreased the induction of the Ptgs2 and Areg promoter activity. EMSA and chromatin immunoprecipitation analyses indicated that NFIL3 binds to the promoter region containing the DNA-binding sites of cAMP response element binding protein and CCAAT enhancer binding protein-β. In summary, hCG induction of NFIL3 expression may modulate the process of ovulation and theca-interstitial and granulosa cell differentiation by regulating expression of PTGS2, PGR, AREG, EREG, and HPGD, potentially through interactions with cAMP response element binding protein and CCAAT enhancer binding protein-β on their target gene promoters.

Penile Growth in Response to Human Chorionic Gonadotropin (hCG) Treatment in Patients with Idiopathic Hypogonadotrophic Hypogonadism

Penile growth is under androgenic control. Human chorionic gonadotropin (hCG) has a stimulatory effect on testicular steroidogenesis and penile growth. The purpose of this study was to evaluate the effect of hCG treatment on the gonadal response and penile growth in male idiopathic hypogonadotrophic hypogonadism (IHH) presenting with micropenis. A total of 20 IHH patients who met the criteria for micropenis were included in this study. hCG (1,500-2,000 IU) was administrated intramuscularly, 3 times per week, for 8 weeks. Basic laboratory and hormonal indexes (including serum testosterone and LH levels), penis length (flaccid and stretched), and testicular volume were measured before and 24 weeks after hCG treatment. The patients' mean age was 18.9 years (range, 12 to 24 years). The mean serum testosterone level was significantly increased after hCG treatment (baseline, 2, 4, 12, and 24 weeks: 0.90±1.35 ng/ml, 1.77±1.31 ng/ml, 3.74±2.24 ng/ml, 5.49±1.70 ng/ml, and 5.58±1.75 ng/ml, respectively; p<0.05). Mean penile length also increased significantly 24 weeks after treatment (flaccid length: from 3.39±1.03 cm to 5.14±1.39 cm; stretched length: from 5.41±1.43 cm to 7.45±1.70 cm; p<0.001). Mean testicular volumes increased significantly as well (left: from 5.45 cc to 6.83 cc; right: from 5.53 cc to 7.03 cc). There were no remarkable adverse effects of the hCG treatment. The hCG treatment increased the serum testosterone level, penile length, and testicular volume in IHH patients. Our results suggest that hCG treatment has a beneficial effect on gonadal function and penile growth in patients with IHH presenting with micropenis.

Xenografting of Human Fetal Testis Tissue: A New Approach to Study Fetal Testis Development and Germ Cell Differentiation

background: Abnormal fetal testis development can result in disorders of sex development (DSDs) and predispose to later testicular dysgenesis syndrome (TDS) disorders such as testicular germ cell tumours. Studies of human fetal testis development are hampered by the lack of appropriate model, and intervention systems. We hypothesized that human fetal testis xenografts can recapitulate normal development. methods: Human fetal testes (at 9 weeks, n ¼ 4 and 14 –18 weeks gestation, n ¼ 6) were xenografted into male nude mice for 6 weeks, with or without hCG treatment of the host, and evaluated for normal cellular development and function using immunohistochemistry, triple immunofluorescence and testosterone assay. The differentiation and proliferation status of germ cells within xenografts was quantified and compared with age-matched controls. results: Xenografts showed .75% survival with normal morphology. In the first-trimester xenografts seminiferous cord formation was initiated and in first- and second-trimester grafts normal functional development of Sertoli, Leydig and peritubular myoid cells was demonstrated using cell-specific protein markers. Grafts produced testosterone when hosts were treated with hCG (P ¼ 0.004 versus control). Proliferation of germ cells and differentiation from gonocytes (OCT4 + ) into pre-spermatogonia (VASA + ) occurred in grafts and quantification showed this progressed comparably with age-matched ungrafted controls. conclusions: Human fetal testis tissue xenografts demonstrate normal structure, function and development after xenografting, including normal germ cell differentiation. This provides an in vivo system to study normal human fetal testis development and its susceptibility to disruption by exogenous factors (e.g. environmental chemicals). This should provide mechanistic insight into the fetal origins of DSDs and TDS disorders.

Expression of peroxiredoxin I regulated by gonadotropins in the rat ovary

ObjectivePeroxiredoxins (Prxs) play an important role in regulating cellular differentiation and proliferation in several types of mammalian cells. This report examined the expression of Prx isotype I in the rat ovary after hormone treatment.MethodsImmature rats were injected with 10 IU of pregnant mare's serum gonadotropin (PMSG) to induce the growth of multiple preovulatory follicles and 10 IU of human chorionic gonadotropin (hCG) to induce ovulation. Immature rats were also treated with diethylstilbestrol (DES), an estrogen analogue, to induce the growth of multiple immature follicles. Northern blot analysis was performed to detect gene expression. Cell-type specific localization of Prx I mRNA were detected by in situ hybridization analysis.ResultsDuring follicle development, ovarian Prx I gene expression was detected in 3-day-old rats and had increased in 21-day-old rats. The levels of Prx I mRNA slightly declined one to two days following treatment with DES. A gradual increase in Prx I gene expression was observed in ovaries obtained from PMSG-treated immature rats. Furthermore, hCG treatment of PMSG-primed rats resulted in a gradual stimulation of Prx I mRNA levels by 24 hours (2.1-fold increase) following treatment, which remained high until 72 hours following treatment. In situ hybridization analysis revealed the expression of the Prx I gene in the granulosa cells of PMSG-primed ovaries and in the corpora lutea of ovaries stimulated with hCG for 72 hours.ConclusionThese results demonstrate the gonadotropin and granulosa cell-specific stimulation of Prx I gene expression, suggesting its role as a local regulator of follicle development.

A fertile male patient with Kallmann syndrome and two missense mutations in the KAL1 gene

To present clinical, genetic, biochemical, and molecular findings in a male fertile patient with Kallmann syndrome (KS). Case report. University medical center. A 26-year-old Chinese man. Clinical evaluation, hormone assays, gene mutation research. Endocrinologic evaluation and genetic analysis. The patient had KS as a result of two missense mutations at exon 11 of KAL1 gene, 1690 G>A and 1765 G>A, a G→A transition in codons 514 and 539, which resulted in the replacement of lysine by glutamic acid, respectively. One of them is a novel mutation. Although loss-of-function mutations of the KAL1 gene is associated with the X-linked form of KS, the reproductive capacity remains unidentified in patients with KS with KAL1 gene mutations. Here, we report a male patient who presented the KS phenotype with fertility. The patient's sperm parameters were lower than the reference ranges; however, a son was born through natural conception after 12 months of marriage. Mutation analysis revealed two KAL1 gene mutations. The results provided further support for the hypothesis that patients with KS can achieve normal sexual development and fertility after hCG treatment, and imply that this genotype is more prevalent in reproductive patients with KS than in the other patients with KS and incurable infertility.

MicroRNA Expression Profiles are Altered by Gonadotropins and Vitamin C Status During In Vitro Follicular Growth

MicroRNAs (miRs) are known to repress target genes at posttranscriptional level and play important roles in the maturation of cells. However, the expression profiles of miRs during follicular maturation have not been fully elucidated. This study was designed to investigate the expression profiles of miRs in murine follicles according to human chorionic gonadotropin (hCG) treatment and vitamin C status during in vitro culture. Ovaries were removed from the 12-day-old wild-type and vitamin C-deficient (L-gulonogammalactone oxidase knockout, Gulo-/-) C57BL6 mice. Preantral follicles were isolated and cultured in 20 µL droplets of culture media supplemented with follicle-stimulating hormone and luteinizing hormone (FSH + LH). After their full maturation, follicles were divided into 2 groups: with and without hCG treatment. Real-time polymerase chain reaction (PCR) was performed using oocytes and granulosa cells (G-cells) to evaluate the miRs known to be expressed mainly in the mouse ovary. After the addition of hCG, miR profiles showed divergent changes between oocytes and G-cells. These profiles significantly differed from those of hCG(-) group. Compared to wild type, Gulo-/- mice showed altered miR profiles in matured oocytes and G-cells. Conclusively, hCG supplementation and vitamin C status alter the miR expression profiles in oocytes and G-cells during in vitro growth of murine follicles.

Adiponectin, Resistin, and Glucose Transporters During the Periovulatory Interval in Rhesus Macaques.

Adiponectin and resistin are part of the adipokine family of hormones produced and secreted by adipose tissue. Circulating adiponectin is associated with increased adiposity and can promote glucose uptake, possibly via increased expression and/or activation of glucose transporters. In addition, a role for adiponectin in reproductive function has been put forth to include effects on ovulation and steroidogenesis. Resistin, in contrast, may be more closely linked to inflammation, although there is some evidence that resistin is associated with insulin resistance. The goal of this study was to examine intra-follicular and serum concentrations of adiponectin and resistin during the periovulatory interval in rhesus monkeys undergoing controlled ovarian stimulation (COS), as well as determine the expression of adiponectin receptors during granulosa cell luteinization. Adult females were treated with recombinant human follicle-stimulating hormone for 7 days and follicles were aspirated before (0 hr), 3, 6, 12, and 24 hr after ovulatory bolus of hCG. Follicular fluid and serum levels of adiponectin and resistin were measured by ELISA before (0 hr) and 24 hr after hCG. Adiponectin decreased (p<0.05) 24 hr after hCG treatment while serum levels remained unchanged. In contrast, follicular fluid levels of resistin were unchanged after hCG, while serum concentrations increased 24 hr post-hCG. The mRNA expression of adiponectin and adiponectin receptors (AdipoR) I and II were analyzed using RT-PCR. Adiponectin mRNA was not detectable in granulosa cells at any time point. However, AdipoRI expression was increased transiently 3 hrs after hCG (5.5-fold, p<0.05), while AdipoRII mRNA was elevated 3 and 6 hrs after hCG treatment before returning to 0 hr levels (6- and 23-fold, respectively; p<0.05). Granulosa cells isolated prior to hCG in vivo were cultured with FSH or FSH+hCG +/- adiponectin (0-25 µg/ml). In the presence of FSH only, adiponectin increased progesterone levels (0 µg/ml adiponectin: 1.7 ng/ml ±0.2; 5 µg/ml: 2.0 ± 0.3; 25 µg/ml: 8.9 ± 0.7). Similarly, hCG markedly increase progesterone over FSH alone (7.7-fold; p<0.05), although adiponectin did not further increase progesterone. Adiponectin did not alter the mRNA expression of glucose transporters (GLUT) 1, 2, or 4, but in the presence of hCG, dose-dependently increased GLUT3 mRNA (p<0.05). These data confirm and quantify the presence of adiponectin and resistin in the luteinizing primate follicle, as well as the dynamic expression of adiponectin receptors. Importantly, the lack of granulosa cell expression of adiponectin mRNA indicates a complex origin for follicular adiponectin. A limited local role for adiponectin through its increased receptors may facilitate glucose uptake by luteinizing granulosa cells. Supported in part by NIH HD043358 (CLC) RR00169 (CNPRC) and RR13439 (CAV). (poster)

A Role for Nuclear Factor Interleukin-3 (NFIL3), a Critical Transcriptional Repressor, in Down-Regulation of Periovulatory Gene Expression.

The LH surge triggers dramatic transcriptional changes in genes associated with ovulation and luteinization. The present study investigated the spatiotemporal expression of nuclear factor IL-3 (NFIL3), a transcriptional regulator of the basic leucine zipper transcription factor superfamily, and its potential role in the ovary during the periovulatory period. Immature female rats were injected with pregnant mare's serum gonadotropin, treated with human chorionic gonadotropin (hCG), and ovaries or granulosa cells were collected at various times after hCG. Nfil3 mRNA was highly induced both in intact ovaries and granulosa cells after hCG treatment. In situ hybridization demonstrated that Nfil3 mRNA was highly induced in theca-interstitial cells at 4–8 h after hCG, localized to granulosa cells at 12 h, and decreased at 24 h. Overexpression of NFIL3 in granulosa cells inhibited the induction of prostaglandin-endoperoxide synthase 2 (Ptgs2), progesterone receptor (Pgr), epiregulin (Ereg), and amphiregulin (Areg) and down-regulated levels of prostaglandin E2. The inhibitory effect on Ptgs2 induction was reversed by NFIL3 small interfering RNA treatment. In theca-interstitial cells the expression of hydroxyprostaglandin dehydrogenase 15-(nicotinamide adenine dinucleotide) (Hpgd) was also inhibited by NFIL3 overexpression. Data from luciferase assays demonstrated that NFIL3 overexpression decreased the induction of the Ptgs2 and Areg promoter activity. EMSA and chromatin immunoprecipitation analyses indicated that NFIL3 binds to the promoter region containing the DNA-binding sites of cAMP response element binding protein and CCAAT enhancer binding protein-β. In summary, hCG induction of NFIL3 expression may modulate the process of ovulation and theca-interstitial and granulosa cell differentiation by regulating expression of PTGS2, PGR, AREG, EREG, and HPGD, potentially through interactions with cAMP response element binding protein and CCAAT enhancer binding protein-β on their target gene promoters.

The pregnancy hormones human chorionic gonadotropin and progesterone induce human embryonic stem cell proliferation and differentiation into neuroectodermal rosettes

IntroductionThe physiological signals that direct the division and differentiation of the zygote to form a blastocyst, and subsequent embryonic stem cell division and differentiation during early embryogenesis, are unknown. Although a number of growth factors, including the pregnancy-associated hormone human chorionic gonadotropin (hCG) are secreted by trophoblasts that lie adjacent to the embryoblast in the blastocyst, it is not known whether these growth factors directly signal human embryonic stem cells (hESCs).MethodsHere we used hESCs as a model of inner cell mass differentiation to examine the hormonal requirements for the formation of embryoid bodies (EB's; akin to blastulation) and neuroectodermal rosettes (akin to neurulation).ResultsWe found that hCG promotes the division of hESCs and their differentiation into EB's and neuroectodermal rosettes. Inhibition of luteinizing hormone/chorionic gonadotropin receptor (LHCGR) signaling suppresses hESC proliferation, an effect that is reversed by treatment with hCG. hCG treatment rapidly upregulates steroidogenic acute regulatory protein (StAR)-mediated cholesterol transport and the synthesis of progesterone (P4). hESCs express P4 receptor A, and treatment of hESC colonies with P4 induces neurulation, as demonstrated by the expression of nestin and the formation of columnar neuroectodermal cells that organize into neural tubelike rosettes. Suppression of P4 signaling by withdrawing P4 or treating with the P4-receptor antagonist RU-486 inhibits the differentiation of hESC colonies into EB's and rosettes.ConclusionsOur findings indicate that hCG signaling via LHCGR on hESC promotes proliferation and differentiation during blastulation and neurulation. These findings suggest that trophoblastic hCG secretion and signaling to the adjacent embryoblast could be the commencement of trophic support by placental tissues in the growth and development of the human embryo.

Xenografting of human fetal testis tissue: a new approach to study fetal testis development and germ cell differentiation

BACKGROUNDAbnormal fetal testis development can result in disorders of sex development (DSDs) and predispose to later testicular dysgenesis syndrome (TDS) disorders such as testicular germ cell tumours. Studies of human fetal testis development are hampered by the lack of appropriate model, and intervention systems. We hypothesized that human fetal testis xenografts can recapitulate normal development.METHODSHuman fetal testes (at 9 weeks, n = 4 and 14–18 weeks gestation, n = 6) were xenografted into male nude mice for 6 weeks, with or without hCG treatment of the host, and evaluated for normal cellular development and function using immunohistochemistry, triple immunofluorescence and testosterone assay. The differentiation and proliferation status of germ cells within xenografts was quantified and compared with age-matched controls.RESULTSXenografts showed >75% survival with normal morphology. In the first-trimester xenografts seminiferous cord formation was initiated and in first- and second-trimester grafts normal functional development of Sertoli, Leydig and peritubular myoid cells was demonstrated using cell-specific protein markers. Grafts produced testosterone when hosts were treated with hCG (P = 0.004 versus control). Proliferation of germ cells and differentiation from gonocytes (OCT4+) into pre-spermatogonia (VASA+) occurred in grafts and quantification showed this progressed comparably with age-matched ungrafted controls.CONCLUSIONSHuman fetal testis tissue xenografts demonstrate normal structure, function and development after xenografting, including normal germ cell differentiation. This provides an in vivo system to study normal human fetal testis development and its susceptibility to disruption by exogenous factors (e.g. environmental chemicals). This should provide mechanistic insight into the fetal origins of DSDs and TDS disorders.

Low-dose human chorionic gonadotropin versus estradiol/progesterone luteal phase support in gonadotropin-releasing hormone agonist–triggered assisted reproductive technique cycles: understanding a new approach

It remains unclear how GnRH agonist (GnRHa) triggering affects the luteal phase, so we investigated the luteal phase after GnRHa triggering, supported with conventional E(2)/P with or without low-dose hCG. E(2)/P support, compared with low-dose hCG, induced a shorter luteal phase (11.2 ± 1.1 vs. 15.0 ± 1.6 days) and fewer subjective complaints (0 vs. 42%), whereas hCG caused more free fluid accumulation and enlarged ovaries than E(2)/P alone. Steroids and low-dose hCG differentially affected corpus luteum function, ovarian size, free fluid accumulation, and patient comfort.

Effect of hCG on Early Luteal Serum Progesterone Concentrations in PG600‐Treated Gilts

Gilt oestrus and ovulation responses to injection of a combination of equine chorionic gonadotrophin (eCG) and human chorionic gonadotrophin (hCG) (PG600) can be unpredictable, possibly reflecting inadequate circulating LH activity. The objective of this study was to determine the effect of PG600 followed by supplemental hCG on gilt ovarian responses. In experiment 1, 212 Hypor gilts (160 day of age) housed on two farms in Spain received intramuscular (i.m.) injections of PG600 (n = 47), or PG600 with an additional 200 IU hCG injected either concurrently (hCG-0; n = 39), or at 24 h (hCG-24; n = 41) or 48 h (hCG-48; n = 45) after PG600. A further 40 gilts served as non-injected controls. Ovulation responses were determined on the basis of initial blood progesterone concentrations being <1 ng/ml and achieving >5 ng / ml 10 d after the PG600 injection. The incidence of ovulating gilts having progesterone concentrations >30 ng/ml were recorded. During the study period, 10% of control gilts ovulated whereas 85-100% of hormone-treated gilts ovulated. There were no significant differences among hormone groups for proportions of gilts ovulating. The proportions of gilts having circulating progesterone concentrations >30 ng/ml were increased (p < or = 0.02) in all hCG treated groups compared with the PG600 group. In experiment 2, a total of 76 Hypor gilts at either 150 or 200 days of age were injected with PG600 (n = 18), 400 IU eCG followed by 200 IU hCG 24 h later (n = 20), PG600 followed by 100 IU hCG 24 h later (n = 17), or 400 IU eCG followed by 300 IU hCG 24 h later (n = 21). Blood samples were obtained 10 days later for progesterone assay. There were no effects of treatment or age on incidence of ovulation, but fewer 150-day-old gilts treated with PG600 or 400 IU eCG followed by 200 IU hCG had progesterone concentrations >30 ng / ml. We conclude that hCG treatment subsequent to PG600 treatment will generate a higher circulating progesterone concentration, although the effect is not evident in older, presumably peripubertal, gilts. The mechanism involved and implications for fertility remain to be determined.

Molecular Mechanisms Mediating the Effect of Mono-(2-Ethylhexyl) Phthalate on Hormone-Stimulated Steroidogenesis in MA-10 Mouse Tumor Leydig Cells

Di-(2-ethylhexyl) phthalate, a widely used plasticizer, and its active metabolite, mono-(2-ethylhexyl) phthalate (MEHP), have been shown to exert adverse effects on the reproductive tract in developing and adult animals. As yet, however, the molecular mechanisms by which they act are uncertain. In the present study, we address the molecular and cellular mechanisms underlying the effects of MEHP on basal and human chorionic gonadotropin (hCG)-stimulated steroid production by MA-10 Leydig cells, using a systems biology approach. MEHP induced dose-dependent decreases in hCG-stimulated steroid formation. Changes in mRNA and protein expression in cells treated with increasing concentrations of MEHP in the presence or absence of hCG were measured by gene microarray and protein high-throughput immunoblotting analyses, respectively. Expression profiling indicated that low concentrations of MEHP induced the expression of a number of genes that also were expressed after hCG stimulation. Cross-comparisons between the hCG and MEHP treatments revealed two genes, Anxa1 and AR1. We suggest that these genes may be involved in a new self-regulatory mechanism of steroidogenesis. The MEHP-induced decreases in hCG-stimulated steroid formation were paralleled by increases in reactive oxygen species generation, with the latter mediated by the Cyp1a1 gene and its network. A model for the mechanism of MEHP action on MA-10 Leydig cell steroidogenesis is proposed.