Ovarian VEGF(165)b expression regulates follicular development, corpus luteum function and fertility.

Angiogenesis and vascular regression are critical for the female ovulatory cycle. They enable progression and regression of follicular development, and corpora lutea formation and regression. Angiogenesis in the ovary occurs under the control of the vascular endothelial growth factor-A (VEGFA) family of proteins, which are generated as both pro-(VEGF(165)) and anti(VEGF(165)b)-angiogenic isoforms by alternative splicing. To determine the role of the VEGF(165)b isoforms in the ovulatory cycle, we measured VEGF(165)b expression in marmoset ovaries by immunohistochemistry and ELISA, and used transgenic mice over-expressing VEGF(165)b in the ovary. VEGF(165)b was expressed in the marmoset ovaries in granulosa cells and theca, and the balance of VEGF(165)b:VEGF(165) was regulated during luteogenesis. Mice over-expressing VEGF(165)b in the ovary were less fertile than wild-type littermates, had reduced secondary and tertiary follicles after mating, increased atretic follicles, fewer corpora lutea and generated fewer embryos in the oviduct after mating, and these were more likely not to retain the corona radiata. These results indicate that the balance of VEGFA isoforms controls follicle progression and luteogenesis, and that control of isoform expression may regulate fertility in mammals, including in primates.

Contrast imaging ultrasound detects abnormalities in the marmoset ovary.

The development of a functional vascular tree within the primate ovary is critical for reproductive health. To determine the efficacy of contrast agents to image the microvascular environment within the primate ovary, contrast ultrasonography was performed in six reproductive-aged female common marmosets (Callithrix jacchus) during the late luteal phase of the cycle, following injection of Sonovue™. Regions of interest (ROIs), representing the corpus luteum (CL) and noncorpus luteum ovarian tissue (NCLOT), were selected during gray-scale B-mode ultrasound imaging. The magnitude of backscatter intensity of CL and NCLOT ROIs were calculated in XnView, post hoc: subsequent gamma-variate modeling was implemented in Matlab to determine perfusion parameters. Histological analysis of these ovaries revealed a total of 11 CL, nine of which were identified during contrast ultrasonography. The median enhancement ratio was significantly increased in the CL (5.54AU; 95% CI -2.21-68.71) compared to the NCLOT (2.82AU; 95% CI 2.73-15.06; P < 0.05). There was no difference in time parameters between the CL and NCLOT. An additional avascular ROI was identified in the ovary of Animal 5, both histologically and by ultrasonography. This cystic ROI displayed a markedly lower enhancement ratio (0.79AU) and higher time parameters than mean CL and NCLOT, including time to peak and time to wash out. These data demonstrate, for the first time, the ability of commercially available contrast agents, to differentiate structures within the nonhuman primate ovary. Contrast-enhanced ultrasonography has a promising future in reproductive medicine.

Molecular mechanisms of ovarian hyperstimulation syndrome: paracrine reduction of endothelial claudin 5 by hCG in vitro is associated with increased endothelial permeability.

BACKGROUND: Ovarian hyperstimulation syndrome (OHSS) is a potentially life-threatening complication of ovarian stimulation associated with severe vascular hyperpermeability. Primary co-cultures of human luteinized granulosa cells (LGCs) and human umbilical vein endothelial cells (HUVECs) were used as a model of steroidgenic/endothelial cell interaction in OHSS. METHODS: hCG and the vascular endothelial growth factor (VEGF) inhibitor, Flt-1Fc, were added to co-cultures of LGCs and HUVECs separated by a micropore membrane. Endothelial permeability to labeled bovine serum albumin was measured and the expression of the endothelial cell-specific adhesion protein claudin 5 was investigated using immunocytochemistry and western blotting. RESULTS: The addition of hCG increased HUVEC permeability in the presence of LGCs (P < 0.05). hCG increased VEGF concentrations in both chambers of the co-culture system (P < 0.05). The increased permeability in the presence of LGCs and hCG was inhibited when VEGF was blocked by Flt-1Fc (P < 0.05). Endothelial membrane claudin 5 protein was reduced in the presence of hCG and LGCs, as measured by immunocytochemistry (P < 0.05) and western blotting (P < 0.05) and this reduction was inhibited by Flt-1Fc. hCG had no direct effects on endothelial cell claudin 5. CONCLUSIONS: For OHSS, this novel paradigm suggests that hCG can increase endothelial permeability by up-regulating VEGF in LGCs which causes reduction in endothelial claudin 5 expression.

Germ cell differentiation in the marmoset (Callithrix jacchus) during fetal and neonatal life closely parallels that in the human.

BACKGROUND: Testicular germ cell tumours (TGCT) are thought to originate from fetal germ cells that fail to differentiate normally, but no animal model for these events has been described. We evaluated the marmoset (Callithrix jacchus) as a model by comparing perinatal germ cell differentiation with that in humans. METHODS: Immunohistochemical profiling was used to investigate germ cell differentiation (OCT4, NANOG, AP-2gamma, MAGE-A4, VASA, NANOS-1) and proliferation (Ki67) in fetal and neonatal marmoset testes in comparison with the human and, to a lesser extent, the rat. RESULTS: In marmosets and humans, differentiation of gonocytes into spermatogonia is associated with the gradual loss of pluripotency markers such as OCT4 and NANOG, and the expression of germ cell-specific proteins such as VASA. This differentiation occurs asynchronously within individual cords during fetal and early postnatal life. This contrasts with rapid and synchronous germ cell differentiation within and between cords in the rat. Similarly, germ cell proliferation in the marmoset and human occurs throughout perinatal life, in contrast to rats in which proliferation ceases during this period. CONCLUSIONS: The marmoset provides a good model for normal human germ cell differentiation and proliferation. The perinatal marmoset may be a useful model in which to establish factors that lead to failure of normal germ cell differentiation and the origins of TGCT.

Regulation of endothelial proliferation by the renin-angiotensin system in human umbilical vein endothelial cells.

This study was performed in order to evaluate the role of angiotensin II in physiological angiogenesis. Human umbilical vein endothelial cells (HUVEC) were stained for angiotensin II type 1 receptor (AGTR1) immunocytochemically and for gene expression of renin-angiotensin system (RAS) components. The regulation of the angiogenesis-associated genes vascular endothelial growth factor (VEGF) and angiopoietins (ANGPT1 and ANGPT2) were studied using quantitative RT-PCR. Furthermore, we examined the effect of angiotensin II on the proliferation of HUVEC using Ki-67 as well as BrdU immunocytochemistry and investigated whether the administration of the AGTR1 blocker candesartan or the VEGF antagonist FLT1-Fc could suppress the observed angiotensin II-dependent proangiogenic effect. AGTR1 was expressed in HUVEC and the administration of angiotensin II significantly increased the gene expression of VEGF and decreased the gene expression of ANGPT1. Since the expression of ANGPT2 was not affected significantly the ratio of ANGPT1/ANGPT2 was decreased. In addition, a significantly increased endothelial cell proliferation was observed after stimulation with angiotensin II, which was suppressed by the simultaneous administration of candesartan or the VEGF antagonist FLT1-Fc. These results indicate the potential capacity of angiotensin II in influencing angiogenesis by the regulation of angiogenesis-associated genes via AGTR1. Since VEGF blockade opposed the effect of angiotensin II on cell proliferation, it is hypothesised that VEGF mediates the angiotensin II-dependent effect in concert with the changes in angiopoietin expression. This is the first report of the RAS on the regulation of angiogenesis-associated genes in physiology.

Potential role of Renin-Angiotensin-system for tumor angiogenesis in receptor negative breast cancer.

OBJECTIVE: This study examined the potential role of Angiotensin II for the regulation of angiogenesis associated genes in receptor positive and negative human breast cancer. METHODS: Expression of different Renin-Angiotensin system (RAS) components in human breast cancer tissue was investigated using immunofluorescence, and in a receptor positive (MCF-7) and receptor negative (MDA-MB 468) breast cancer cell line by performing immunocytochemistry and RT-PCR. Both cell lines were stimulated with Angiotensin II and Angiotensin II receptor type 1 (At(1)R) blocker Candesartan, and gene expression of vascular endothelial growth factor (VEGF), Angiopoietin 1 and 2 (Ang-1 and Ang-2), tissue inhibitor of matrix metalloproteinases 1 (TIMP-1), and hypoxia inducible transcription factor 2alpha (HIF-2alpha) were quantified by TaqMan-Real-Time PCR analysis. RESULTS: RAS components, Angiotensinogen, Renin, Angiotensin I-converting enzyme (ACE), and At(1)R and At(2)R were expressed in hormone-receptor negative and positive human breast cancer tissue as well as in MDA-MB 468 and in MCF-7 human breast cancer cells. In addition, we found expression of VEGF, Ang-1, TIMP-1, and HIF-2alpha in both cell lines. However, only in receptor negative MDA-MB 468 cells, did Angiotensin II significantly increase gene expression of VEGF, HIF-2alpha, and TIMP-1. This effect was completely inhibited by Candesartan. CONCLUSION: In conclusion, it is hypothesized that Angiotensin II may be involved in regulation of tumor angiogenesis especially in receptor negative breast cancer by regulation of angiogenesis associated genes via At(1)R. These findings are the first evidence for targeting tumor angiogenesis by inhibition of At(1)R in receptor negative human breast cancer cells and may lead to new therapeutical anticancer strategies based upon inhibition of At(1)R.

Regulation of tight junction proteins occludin and claudin 5 in the primate ovary during the ovulatory cycle and after inhibition of vascular endothelial growth factor.

Ovarian follicular and corpus luteum development, including angiogenesis, are characterized by cell-cell rearrangements that may require dynamic changes in cell-cell adhesion. The present study investigates the expression of tight junction proteins occludin and claudin 5 during follicular and luteal development in the primate ovary and after inhibition of vascular endothelial growth factor (VEGF) by VEGF trap treatment. Occludin was localized to the plasma membrane of granulosa cells. During follicular development occludin staining decreased significantly (P < 0.05) and disappeared completely by the ovulatory stage. After inhibition of VEGF, occludin staining was significantly (P < 0.05) higher in the granulosa of secondary and tertiary follicles compared with controls. Claudin 5 was exclusively localized to the theca vasculature. A significant (P < 0.05) increase in staining was detected from the pre-antral to the antral and ovulatory stage. However, dual staining with CD31 revealed that within the theca endothelium the amount of claudin 5 remained constant during follicular development. Treatment with VEGF trap throughout the follicular phase revealed a lack of claudin 5 staining in the theca interna but no difference was observed in the remaining theca externa vasculature. In the corpus luteum, claudin 5 was also localized in the vasculature. Treatment with VEGF trap in the mid-luteal phase resulted in a significant increase in staining (P < 0.05). These results led us to hypothesize that tight junctions are involved in regulation of follicular growth, antrum transition and follicular angiogenesis which is compromised by VEGF inhibition. VEGF may influence luteal vascular permeability by regulation of the endothelial specific tight junction protein claudin 5.

Control of the preovulatory Luteinizing Hormone surge by Gonadotropin-Releasing Hormone antagonists: prospects for clinical application.

The preovulatory LH surge of the primate menstrual cycle represents a number of positive influences, a major component of which is a direct action of estradiol on the anterior pituitary lobe. Whether the LH surge also requires a corresponding burst of GnRH release from the hypothalamus has been debated. After many years of investigation, there is now conclusive evidence that a midcycle GnRH surge does occur in the primate. This is supported by studies in women with normal ovulatory cycles that demonstrate that blockade of the GnRH receptor by potent GnRH antagonists administered within 1-2 days of the expected midcycle can delay the LH surge. The ability to prevent the positive feedback effects of estradiol by GnRH antagonists is being employed for the controlled induction of follicular development and ovulation in the treatment of infertility and in in vitro fertilization programs.

Does inhibin have an endocrine function during the menstrual cycle?

Inhibin (alpha-beta heterodimer) has been considered to be the principal nonsteroidal ovarian regulator of pituitary FSH secretion. The beta-beta heterodimer, activin, produced by the ovary and other tissues, appears to act locally, with actions opposite to those of inhibin. Since immunoreactive inhibin is highest during the luteal phase of the menstrual cycle when FSH is lowest, a negative feedback role in controlling FSH release at this time has been suggested. Attempts to establish this by using immunoneutralization techniques have failed to reveal such a role. We must enhance our understanding of the gonadotropic control of inhibin/activin gene expression within the various compartments of the primate ovary, the role of their binding proteins, and the nature of the secretory products before we can resolve the question of whether inhibin has an endocrine function during the menstrual cycle and how cyclic reinitiation of follicular development is controlled.

Compensatory response of the luteinizing-hormone (LH)-releasing hormone (LHRH)/LH pulse generator after administration of a potent LHRH antagonist in the ram.

It is established that the blockade of the pituitary LHRH receptor by an LHRH antagonist will suppress pituitary LH secretion and reduce serum concentrations of gonadal steroids. Little is known, however, about the activity of the LHRH/LH pulse generator during this inhibitory period or during the recovery phase. To investigate this, a potent LHRH antagonist [N-Ac-D-pCl-Phe1,D-pCl-Phe2,D-Trp3,D-hArg(Et2)6, D-Ala10 LHRH was injected iv into sexually active rams and the changes in the blood plasma concentrations of LH, FSH, testosterone, and PRL were measured in samples collected every 15 min for 24-48 h. The treatment induced an immediate blockade of pulsatile LH secretion and a parallel decline in blood levels of testosterone. Plasma levels of FSH were not suppressed by treatment with the LHRH antagonist and there was no consistent effect on plasma levels of PRL. The duration of the inhibition of LH was dose dependent lasting 4.3 +/- 0.4 h, 18.0 +/- 1.0 h, and 31.8 +/- 1.3 h for the low (6 micrograms/kg), medium (36 micrograms/kg), and high (365 micrograms/kg) doses of LHRH antagonist, respectively. During the recovery period there was an approximate 2-fold increase in the frequency of LH pulses. These results suggest a compensatory response to the decline in the negative feedback effect of testosterone secretion. Even the lowest dose of antagonist elicited a decrease in the level of testosterone and an increase in LH pulse frequency. At this dose, the decline in testosterone was very transitory indicating an acute sensitivity of the hypothalamus to changes in the negative feedback signal. These results suggest that the suppression of LH and testosterone secretion in the ram by LHRH antagonist is associated with a compensatory increase in the activity of the LHRH pulse generator.

Increased sensitivity to the negative feedback effects of testosterone induced by hyperprolactinemia in the adult male rat.

High plasma levels of PRL induced by transplants of two donor pituitaries under the kidney capsule of adult male rats resulted in a prolonged suppression of plasma levels of LH and FSH although testosterone levels were maintained within normal limits. Castration of rats with pituitary transplants resulted in a normal though delayed rise in serum levels of both LH and FSH to levels equivalent to those in normal castrated controls. This increase in gonadotropin levels occurred in spite of maintenance of elevated PRL levels. Two experiments were carried out in which testosterone was restored after castration by Silastic testosterone-containing implants of various lengths (Exp 1:60, 30, and 10 mm; Exp 2: 30, 20, 10, 5, and 2 mm). In both experiments 60- and 30-mm testosterone implants prevented the postcastration rise in LH and FSH in both control and hyperprolactinemic rats. However, although the shorter testosterone implants delayed this rise in control rats, levels of LH and FSH increased by 4 days and were not significantly different from castrated rats without testosterone implants by 15 days after castration. In contrast, this rise in gonadotropins was abolished or considerably delayed by the shorter implants in hyperprolactinemic rats, demonstrating an increase in sensitivity of the hypothalamic pituitary axis to the negative feedback effects of testosterone in these animals. These results suggest that 1) to maintain suppression of gonadotropin secretion in hyperprolactinemia high levels of PRL alone are insufficient and gonadal steroids are required, and 2) high levels of PRL appear to sensitize the hypothalamic-pituitary axis to the negative feedback effects of gonadal steroids.

Suppression of luteal function by a luteinizing hormone-releasing hormone antagonist during the early luteal phase in the stumptailed macaque monkey and the effects of subsequent administration of human chorionic gonadotropin.

In previous studies a single sc injection of the LHRH antagonist [N-Ac-D-Nal(2)1,D-pCl-Phe2,D-Trp3,D-hArg(Et2)6,D-Ala10 ]LHRH during the luteal phase of the stumptailed macaque menstrual cycle caused a transient suppression of serum LH and progesterone concentrations. To investigate whether a more prolonged suppression of LH release during the early luteal phase could result in a sustained suppression of progesterone, 10 monkeys were treated with 3 consecutive daily injections of 300 micrograms LHRH antagonist/kg beginning on days 0 (n = 2), 1 (n = 1), 2 (n = 1), 3 (n = 2), 4 (n = 2), and 5 (n = 2) after the LH surge. When the antagonist was administered on the day of the LH surge, serum concentrations of bioactive LH were still elevated on the following day, but then fell to low levels. Serum progesterone concentrations were subnormal in these monkeys for the next 10 days, but recovered toward the late luteal phase. In the 8 monkeys receiving antagonist starting between days 1-5 after the LH surge, serum concentrations of bioactive LH were suppressed to near the detection limit of the assay for 4 days after the first injection. Seven of the 8 monkeys demonstrated a progressive decline in serum progesterone concentrations to undetectable values which remained for the duration of the luteal phase. In the remaining monkey the decline in progesterone was less marked; this animal presented a normal progesterone profile 3 days after the last antagonist injection. Premature menses occurred in all 8 monkeys; the next ovulation occurred 18.9 +/- 0.3 days after the last antagonist injection. To test luteal function after antagonist treatment during the early luteal phase and to mimic the rescue of the corpus luteum during a fertile cycle and assess the contraceptive effects of antagonist, hCG in daily doses of 30, 60, 90, 180, and 360 IU was administered starting on day 7 of the luteal phase to monkeys previously treated with three daily injections of 300 micrograms antagonist/kg during the early luteal phase. Control monkeys received hCG injections alone. In the controls, hCG administration elevated serum progesterone concentrations to 15-20 ng/ml. In three monkeys in which antagonist administration did not commence until day 5 or 6, hCG overcame the suppressive effect of the antagonist. However, in seven monkeys in which antagonist administration began on days 1-4, hCG caused only a small progesterone rise (maximal range, 1.8-4.9 ng/ml), about 20% of that observed in control monkeys receiving hCG.(ABSTRACT TRUNCATED AT 400 WORDS)

Thyrotropin-releasing hormone in rat pituitary stalk blood and hypothalamus: studies with high performance liquid chromatography.

Immunoreactive TRH (TRH-IR) in rat pituitary stalk blood and hypothalamus was investigated with high performance liquid chromatography in conjunction with a sensitive RIA. The TRH-IR of ethanol extracts of stalk blood resolved into three peaks, whether the blood was collected with the pituitary gland in situ or after the gland had been removed. The first peak corresponded to authentic TRH. By contrast, all the immunoreactive TRH in pituitary and hypothalamic extracts and in peripheral blood to which hypothalamic or synthetic TRH had been added eluted as a single peak with the same retention time as authentic TRH. The additional IR peaks in stalk blood did not correspond to known metabolites of TRH, and, since absent from the hypothalamus, they are unlikely to represent stored TRH precursors. Although authentic TRH constituted only 37% of total TRH-IR in pituitary stalk blood, the amount released during the first hour into stalk blood (1.7 ng) in relation to hypothalamic content (approximately 5 ng) was still high (approximately 34%) compared with that of LHRH (approximately 0.6%) and somatostatin (approximately 0.3%).

Cloning and functional expression of the luteinizing hormone receptor complementary deoxyribonucleic acid from the marmoset monkey testis: absence of sequences encoding exon 10 in other species.

Based on sequence homologies among the human, porcine, rat, and mouse genes for the LH receptor (LHR), overlapping partial fragments of LHR complementary DNAs (cDNAs) were multiplied from marmoset monkey testicular RNA using reverse transcription-PCR. Ligations of the individual cDNA fragments generated a full-length monkey LHR cDNA (2031 bp) containing the complete amino acid-coding sequence (676 amino acids). Northern hybridization analysis of monkey testicular RNA, using a complementary RNA probe corresponding to the full-length cDNA, demonstrated major transcripts of 5.5 and 1.4 kilobases and minor ones of 4.0, 2.7, and 1.9 kilobases. Sequence analysis of the monkey LHR cDNA revealed a striking feature, i.e. the absence of an 81-bp nucleotide sequence corresponding to exon 10, present in the LHR cDNAs of all other species studied to date. The monkey LHR cDNA displayed 83-94% overall sequence homology with the other mammalian LHR cDNAs. Reverse transcription-PCR with human exon 10-specific primers demonstrated the total absence of this sequence from the monkey LHR messenger RNA. Southern hybridization of monkey genomic DNA using a human exon 10 probe demonstrated its presence in the monkey gene and that it is totally spliced out from the primary transcript. COS cells transfected with the monkey LHR cDNA showed similar high affinity (Kd = 0.25 nmol/liter) of [125I]iodo-hCG binding as those transfected with human LHR cDNA (Kd = 0.20 nmol/liter). The cells expressing the recombinant monkey and human LHR displayed similar responses of extracellular cAMP and inositol trisphosphate to hCG. In conclusion, marmoset monkey LHR seems to lack the sequence corresponding to exon 10 of the LHR gene in other mammalian species. The truncation does not alter LHR function, as the monkey receptor protein bound hCG and evoked cAMP and inositol trisphosphate responses comparable to those of the human LHR containing the exon 10-encoded structure. As the sequence homologous to exon 10 is missing in the other two glycoprotein receptors, i.e. those of FSH and TSH, this extra exon is apparently inserted into the LHR messenger RNA of some species during evolution from intronic sequences by a change in alternative splicing.

Angiogenesis during follicular development in the primate and its inhibition by treatment with truncated Flt-1-Fc (vascular endothelial growth factor Trap(A40)).

The aims of this study were to 1) quantify changes in angiogenesis during follicular growth in a primate model; 2) investigate the molecular regulation using in situ hybridization of vascular endothelial growth factor (VEGF), its receptor, Flt-1, the angiopoietins (Ang-1 and Ang-2), and their receptor, Tie-2; 3) elucidate the role of VEGF in follicular angiogenesis by blocking its action by treatment with a soluble truncated form of the Flt-1 receptor, (VEGF Trap(A40)). Changes in angiogenesis were quantified using bromodeoxyuridine to obtain a proliferation index, and CD31 immunocytochemistry to visualize endothelial cell area. Percentage of proliferating endothelial cells was calculated by double labeling for bromodeoxyuridine and CD31. Vascularization was first observed in follicles containing four granulosa cell layers. A significant increase in proliferation in the thecal layer was observed from the early to late secondary stage, and dual staining showed that 25% of proliferating cells were of endothelial cell origin. VEGF messenger RNA (mRNA) was expressed in granulosa cells with an increase of grain density from late secondary to tertiary follicles. Ang-1 was weakly expressed in the theca of tertiary follicles. Ang-2 mRNA was not detected in any follicles. The mRNA for the Flt-1 and Tie-2 receptors was localized in endothelial cells of the theca. Unexpectedly, Tie-2 mRNA was also found in granulosa cells of early follicular stages and its translation was confirmed by immunocytochemistry. VEGF trap treatment for 3 days resulted in an 87% decrease of proliferation in the theca of secondary and tertiary follicles, a reduction in endothelial cell area and a marked decline in Flt-1 mRNA expression. Granulosa cell proliferation also decreased. These results show that onset and establishment of the follicle vasculature takes place early during follicular development. The ability of VEGF trap treatment to severely restrict follicular angiogenesis establishes that VEGF is the major regulator of this process in the primate ovary.

Testosterone regulation of proopiomelanocortin messenger ribonucleic acid in the arcuate nucleus of the male rat.

GnRH regulates the secretion of LH and FSH, which stimulate the secretion of testicular hormones. Acting in a reciprocal fashion, these hormones, including testosterone and inhibin, exert a negative feedback effect on GnRH and gonadotropin secretion. Endogenous opioid peptides (EOPs) have been implicated to play a role in steroid-mediated regulation of gonadotropin secretion. In this context, certain steroid hormones (e.g. testosterone) increase EOP activity and ultimately inhibit GnRH secretion; however, the cellular mechanism by which this occurs is unknown. beta-Endorphin is one of these EOPs, and it is derived from a larger precursor molecule, POMC. We tested the hypothesis that testicular hormones and testosterone, in particular, stimulate POMC gene expression in the arcuate nucleus of the male rat brain. First, we compared POMC mRNA levels between intact and castrated male rats. Adult male rats were killed 4 days (n = 4) and 21 days (n = 5) after castration. Intact animals (sham-operated; n = 6) were used as controls. Using in situ hybridization and a computerized image analysis system, we measured the POMC mRNA content in individual cells of the arcuate nucleus. POMC mRNA signal was significantly lower (P less than 0.0003) in both 4-day (126 +/- 2 grains/cell) and 21-day (117 +/- 5 grains/cell) castrates than in controls (142 +/- 2 grains/cell). In a second experiment we tested whether testosterone would reverse the castration-induced loss of POMC message. Again, we castrated animals and immediately implanted them with either empty (sham; n = 6) or testosterone-containing Silastic implants (n = 5) of a size that would deliver physiological levels of testosterone (3.6 +/- 1.5 ng/ml). We observed that testosterone-treated animals had significantly higher levels of POMC mRNA signal (121.8 +/- 3.8 grains/cell) than sham-treated castrates (111.4 +/- 3.6 grains/cell; P less than 0.03) and that the testosterone-treated castrates had POMC mRNA signal levels indistinguishable from those of intact controls (122.0 +/- 1.1 grains/cell). These observations lend credence to the theory that one mechanism by which testosterone may regulate GnRH secretion is by increasing the synthesis of POMC in the arcuate nucleus.

Immunoexpression of aquaporin-1 in the efferent ducts of the rat and marmoset monkey during development, its modulation by estrogens, and its possible role in fluid resorption.

Recent data suggest that estrogens play a role in regulating fluid resorption from the efferent ducts, though the biochemical mechanisms involved are unknown. The present study has used immunocytochemistry to localize a water channel protein, Aquaporin-1 (AQP-1), to the efferent ducts of male rats and marmoset monkeys from perinatal life through to adulthood and has then investigated its potential hormonal regulation in neonatal/peripubertal life, via administration of a GnRH antagonist (GnRHa) or diethylstilbestrol (DES) to rats. AQP-1 was immunoexpressed intensely in the apical brush border of the epithelium lining the efferent ducts at all ages studied, from late fetal life through puberty to adulthood. In the marmoset, but not the rat, AQP-1 was also expressed in the epithelium of the rete testis. Once the cell types within the efferent duct epithelium had differentiated, it was clear that only nonciliated cells of the rat localized AQP-1. When gonadotropin secretion was suppressed in rats by neonatal administration of GnRHa, immunoexpression of AQP-1 at age 18 and 25 days was virtually unchanged in intensity, though the efferent ducts were reduced in size. In contrast, when DES was administered neonatally to rats (up to day 12), immunoexpression of AQP-1 was reduced at day 10, virtually abolished at day 18, reduced markedly at day 25 and to a small extent at day 35; these findings were confirmed by Western blot analysis at day 18. The DES-induced decrease in immunoexpression of AQP-1 was accompanied by pronounced distension of the efferent ducts and rete, consistent with reduced fluid resorption. The epithelial cells of the efferent ducts in DES-treated rats were cuboidal rather than columnar in shape as in controls and were reduced significantly in height compared with controls at all ages through to adulthood. These findings suggest that estrogens may play a role in regulating fluid resorption from the efferent ducts during fetal/neonatal development and/or a role in the gross and functional development of the efferent ducts and rete testis. The present data also suggest that AQP-1 is one of the elements involved in the regulation of fluid resorption in the efferent ducts. The importance of fluid flow in fetal/neonatal development of the excurrent duct system of the male is also suggested by these observations.

Suppression of luteal angiogenesis in the primate after neutralization of vascular endothelial growth factor.

Manipulation of angiogenesis may have a profound effect on female reproductive function, but this has not yet been demonstrated by direct experiment in species with ovulatory cycles similar to those in women. To investigate whether angiogenesis could be inhibited in the primate corpus luteum, and the consequences of such inhibition on luteal function, marmosets were treated with an antibody to vascular endothelial growth factor (VEGF). Treatment commenced at the time of ovulation and was continued for 3 days (early luteal group) or 10 days (midluteal group). Bromodeoxyuridine was used to label proliferating cells, being administered 1 h before collecting ovaries from control and treated animals in the early or midluteal phase. Ovarian sections were stained using an antibody to bromodeoxyuridine, and a proliferation index was obtained; endothelial cell quantification was performed using factor VIII as an endothelial cell marker. Intense proliferation in the early luteal phase was suppressed by anti-VEGF treatment. This resulted in blockade of development of the normally extensive capillary bed, as in the animals treated until the mid-luteal phase the numbers of endothelial cells were reduced. The hormone-producing cells remained largely unaltered in the posttreatment corpus luteum, although the presence of lipid accumulation, and small pockets of cells showing basophilia and nuclear condensation were observed. Significantly, luteal function, as judged by secretion of progesterone, was markedly compromised by the treatment, being reduced by 60% in comparison with controls. It is concluded that VEGF-mediated angiogenesis is an essential component of luteal function in primates and therefore has the potential to be regulated.

Suppression of follicular maturation by infusion of a luteinizing hormone-releasing hormone agonist starting during the late luteal phase in the stumptailed macaque monkey.

The effect of continuous administration of an LHRH agonist for 14 days on follicular estradiol secretion was studied in adult female stumptailed macaque monkeys. The infusion was started either during the early follicular phase or during the late luteal phase. Both treatments resulted in a rise in serum concentrations of LH and FSH for 2-3 days, after which time values returned to basal levels. Infusion during the early follicular phase induced a rapid rise in serum concentrations of estradiol, which declined after 7-10 days. The normal rise in serum progesterone after midcycle failed to occur, indicating that ovulation had been inhibited. Infusion during the late luteal phase of the cycle prevented the normal rise in serum concentrations of FSH and estradiol at the end of the luteal phase, and there was no indication of follicular estradiol production during the subsequent infusion period. Removal of the minipumps after 14 days was followed promptly by normal cycles, regardless of when the infusion had been started. In a second experiment, infusion of LHRH agonist was started during the luteal phase and extended for 90 days. This resulted in marked and sustained suppression of estradiol secretion and total absence of progesterone rises. These findings show that in the monkey, a rapid and sustained suppression of estradiol production can be obtained if a long term infusion of LHRH agonist is started during the luteal phase of the cycle. Such an approach in women using infusion pumps or sustained delivery preparations may be the most effective way of achieving a medical ovariectomy for the treatment of endometriosis, leiomyomata, and other estrogen-dependent pathological conditions.

Inhibition of early luteal angiogenesis by gonadotropin-releasing hormone antagonist treatment in the primate.

Angiogenesis during luteal development is essential for normal lutein cell function, but the control of this process and the relationships between the steroidogenic and endothelial cells have still to be elucidated. The aim of this study was to: 1) quantify endothelial cell proliferation throughout the luteal phase of the marmoset ovulatory cycle; 2) determine the effect of gonadotropin withdrawal using GnRH antagonist treatment on the early luteal phase angiogenesis peak; and 3) describe the resultant morphological changes in the corpus luteum (CL). Ovaries were collected during the early, mid-, and late luteal phase, and changes in angiogenic activity were determined by quantification of bromodeoxyuridine incorporation. Animals were treated with a GnRH antagonist, on luteal days 1 and 2, and ovaries were collected on day 3. A proliferation index was obtained by counting the number of bromodeoxyuridine immunopositive cells in luteal sections. Cell proliferation was maximal in the early luteal phase and fell significantly in the mid- and late CL. GnRH antagonist treatment reduced the early luteal phase proliferation peak by 90%, suppressed plasma progesterone, and severely disrupted lutein cell morphology. These results demonstrate that the intense angiogenesis in the early primate CL is dependent on gonadotropin stimulation of lutein cells.