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.

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.

KiSS-1 in the mammalian ovary: distribution of kisspeptin in human and marmoset and alterations in KiSS-1 mRNA levels in a rat model of ovulatory dysfunction.

Kisspeptins, the products of the KiSS-1 gene acting via G protein-coupled receptor 54 (GPR54), have recently emerged as pivotal signals in the hypothalamic network triggering the preovulatory surge of gonadotropins and, hence, ovulation. Additional actions of kisspeptins at other levels of the hypothalamic-pituitary-ovarian axis have been suggested but remain to date scarcely studied. We report herein the pattern of expression of KiSS-1 and GPR54 in the human and nonhuman primate ovary and evaluate changes in ovarian KiSS-1 expression in a rat model of ovulatory dysfunction. KiSS-1 and GPR54 mRNAs were detected in human ovarian tissue and cultured granulosa-lutein cells. In good agreement, kisspeptin immunoreactivity was observed in cyclic human and marmoset ovaries, with prominent signals in the theca layer of growing follicles, corpora lutea, interstitial gland, and ovarian surface epithelium. GPR54 immunoreactivity was also found in human theca and luteal cells. Administration of indomethacin to cyclic female rats disturbed ovulation and resulted in a dramatic drop in ovarian KiSS-1, but not GPR54, cyclooxygenase-2 (COX-2), or progesterone receptor, mRNA levels at the time of ovulation; an effect mimicked by the selective COX-2 inhibitor NS398 and rescued by coadministration of PGE(2). Likewise, the stimulatory effect of human choriogonadotropin on ovarian KiSS-1 expression was partially blunted by indomethacin. In contrast, KiSS-1 mRNA levels remained unaltered in another model of ovulatory failure, i.e., the RU486-treated rat. In summary, we document for the first time the expression of KiSS-1/kisspeptin and GPR54 in the human and nonhuman primate ovary. In addition, we provide evidence for the ability of inhibitors of COX-2, known to disturb follicular rupture and ovulation, to selectively alter the expression of KiSS-1 gene in rat ovary. Altogether, our results are suggestive of a conserved role of local KiSS-1 in the direct control of ovarian functions in mammals.

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.

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 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.

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.

Effects of inhibition of vascular endothelial growth factor at time of selection on follicular angiogenesis, expansion, development and atresia in the marmoset.

This study determined the effects of inhibiting vascular endothelial growth factor (VEGF) at follicle selection. Marmosets were given an injection of VEGF antagonist, the VEGF Trap on Day 5 of the follicular phase and ovaries were evaluated on Day 10 or 15. Ovaries from controls were assessed on Day 5 (time of selection), Day 10 (peri-ovulatory) and Day 15 (luteal phase). At Day 10, ovaries of four of the five controls contained dominant follicles, while one had ovulated. VEGF Trap-treated ovaries also contained large follicles on Day 10, but VEGF inhibition had suppressed endothelial cell proliferation, leading to reductions in the thecal vascularization and plasma estradiol relative to controls. By Day 15, ovaries of controls contained active corpora lutea whereas ovaries of four of the five treated animals still contained large antral follicles similar in size to pre-ovulatory follicles, and one had small, avascular corpora lutea. However, these follicles had a restricted vasculature, increased incidence of activated caspase-3 staining and morphological features indicating they would become degenerative non-functional cysts. These results show that after follicle selection, VEGF is essential for angiogenesis and the generation of healthy ovulatory follicles and corpora lutea, but fluid accumulation can still occur in selected follicles in the absence of VEGF.

Cell junctional proteins in the human corpus luteum: changes during the normal cycle and after HCG treatment.

BACKGROUND: Regulation of tissue remodelling and ovarian permeability by intercellular adhesion complexes may be involved in normal and pathological ovarian function. Therefore, the occurrence, distribution and hormonal control of the adherens junction protein vascular endothelial cadherin (VE-cadherin) and the tight junction proteins occludin and claudin in the human corpus luteum (CL) were investigated. METHODS: CLs from patients undergoing hysterectomy for benign reasons were enucleated during early, mid- and late stages of the functional luteal phase and after HCG rescue in vivo. Immunostaining for occludin, claudins 1 and 5 and VE-cadherin was carried out on fixed tissue. Endothelial cells, granulosa lutein cells and theca lutein cells were identified by reference to serial sections immunostained for CD34, 17alpha-hydroxylase and 3beta-hydroxy-steroid-dehydrogenase. Quantitative analyses were performed using image analyses. RESULTS: Occludin was localized to the plasma membrane of granulosa lutein cells and endothelial cells but was absent in theca lutein cells. Claudin 1 was exclusively localized to the plasma membrane of steroidogenic cells. Claudin 5 and VE-cadherin were only present in endothelial cells. After HCG administration in vivo, adherens and tight junction proteins were significantly down-regulated (P < 0.05). CONCLUSIONS: The decrease of junctional proteins after HCG treatment suggests a hormonal control of tight and adherens junctions in the CL associated with tissue remodelling and an increase in luteal permeability during early pregnancy.

Temporal relationship between proliferating and apoptotic hormone-producing and endothelial cells in the equine corpus luteum.

The temporal relationship between endothelial cell death, vascular regression and the death of hormone-producing cells in the mare has not been established. To determine the dynamics of cell proliferation and death throughout the luteal phase, corpora lutea were studied at the early, mid- and late luteal phase, and after treatment with cloprostenol in the mid-luteal phase to induce premature luteolysis. Changes in cell proliferation and apoptosis were investigated utilising specific markers (phosphorylated histone-3 and activated caspase-3 respectively). Histone-3 positive cells were most abundant during the early luteal phase, and were mainly present in endothelial cells. Histone-3 activity significantly increased in hormone-producing cells 36 h after cloprostenol treatment. Frequency of activated caspase-3 staining peaked on day 14, and was induced by 36 h after cloprostenol administration in mid-luteal phase. However, cell death occurred simultaneously in the endothelial and hormone-producing cells. These results show that a subset of hormone-producing cells enter the early stages of cell division around luteolysis, while the majority of cells are undergoing cell death. Natural and induced functional and structural luteal regression in the mare can be at least partially attributed to simultaneous apoptosis of endothelial and hormone-producing cells. However, there is no evidence that endothelial cell death is the trigger for naturally occurring luteolysis.

Effects of GnRH antagonist treatment on follicular development and angiogenesis in the primate ovary.

Angiogenesis is required for normal follicular development but the role of gonadotrophins in the control of follicular angiogenesis remains to be elucidated. This study investigated the effects of treatment with GnRH antagonist in vivo on follicular development and angiogenesis in the marmoset. GnRH antagonist was administered on either follicular day 0 or day 5 of the 10-day follicular phase with ovaries collected on day 10. Ovaries from control marmosets were studied at day 5 (mid follicular phase) and day 10 (periovulatory period). Ovaries were fixed, serial sectioned and subjected to morphological analysis and immunocytochemistry to determine cell proliferation and follicular endothelial cell area and in situ hybridization to assess changes in expression of vascular endothelial growth factor (VEGF). Treatment with GnRH antagonist from day 0-10 resulted in an absence of dominant preovulatory follicles seen in controls. In the remaining tertiary follicles granulosa, theca and endothelial cell proliferation was reduced, resulting in a minor reduction in vascular density. However, VEGF mRNA expression was unaffected by treatment. Treatment from day 5-10 did not prevent development of ovulatory size follicles, but they were atretic and lacked VEGF mRNA. These results suggest that while VEGF expression in the preovulatory follicle is under gonadotrophic control it is not dependent on normal gonadotrophin secretion in tertiary follicles, indicating that there are other paracrine factors regulating VEGF expression in the developing ovarian follicle.

Role of the neonatal period of pituitary-testicular activity in germ cell proliferation and differentiation in the primate testis.

BACKGROUND: The neonatal period of pituitary-testicular activity (NPTA) in human males has been hypothesized to play a role in germ cell proliferation and differentiation and to be defective in cryptorchid testes. The present study was carried out to establish in the marmoset if suppression of the NPTA, by treatment with a GnRH antagonist, results in impaired germ cell proliferation and/or differentiation. METHODS: Comparison of germ cell (GC) numbers and differentiation from gonocytes to pre-spermatogonia and spermatogonia, at birth (in controls) and at the end of the NPTA in marmoset co-twin males treated from birth to age 14 weeks with vehicle or GnRH antagonist. RESULTS: From birth to age 18-24 weeks, testis weight increased approximately 5-fold and GC number approximately 10-fold, including increased numbers of gonocytes and pre-spermatogonia and the first appearance of spermatogonia. Treatment with GnRH antagonist attenuated the increase in testis weight and GC numbers, but the effect was only partial (24-30% reduction), and the relative proportions of gonocytes, pre-spermatogonia and spermatogonia in the GnRH antagonist-treated group were unchanged from control values. CONCLUSIONS: The NPTA plays only a minor, if any, role in GC proliferation and differentiation in the marmoset. The changes in GnRH antagonist-treated co-twins may reflect impaired GC survival due to withdrawal of gonadotrophin support for Sertoli cells. These findings do not support a pivotal role for the NPTA in neonatal GC development in primates.

Localization of mRNA for vascular endothelial growth factor (VEGF), angiopoietins and their receptors during the peri-implantation period and early pregnancy in marmosets (Callithrix jacchus).

Implantation of a blastocyst into a receptive endometrium is a prerequisite for successful pregnancy. Angiogenesis is a key event in this process but the mechanisms by which localized changes in vascular permeability and angiogenesis occur have yet to be elucidated. Vascular endothelial growth factor (VEGF) and its receptors VEGFR-1 and VEGFR-2 have been implicated as key players in vascular remodelling and placentation. Angiopoietins also appear to have a significant role in regulation of blood vessel growth, maturation and regression. The aim of this study was to describe the molecular regulation of angiogenesis in the first month of pregnancy in marmosets and to address the putative physiological roles for these factors. Uteri were studied at weeks 2, 3 and 4 of pregnancy and compared with late secretory non-pregnant endometrium. Implantation in marmosets occurs at day 11 of pregnancy; hence, these time points were chosen so that the peri-implantation period and very early pregnancy could be studied. mRNAs for VEGF, VEGFR-1 and VEGFR-2, angiopoietin 1, angiopoietin 2 and their receptor Tie-2 were localized and quantified by in situ hybridization. Endothelial cells were identified by CD31 immunocytochemistry. VEGF mRNA was present in all compartments except endothelial cells, and its expression generally increased throughout pregnancy except in upper zone glandular epithelium and luminal epithelium, where a decrease in expression was observed. VEGF receptor mRNAs were found in endothelial cells of the upper zones immediately surrounding glandular epithelium. Angiopoietin 1 mRNA was localized to glandular epithelium of the upper and lower zones throughout pregnancy, and increased in stroma at week 4. Expression of angiopoietin 2 mRNA was localized exclusively to endothelial cells of large luminal vessels and was higher in endometrium from marmosets at week 4 of pregnancy than in endometrium from all other stages. These data provide comprehensive evidence that VEGFR-1 and -2, and angiopoietin 1, angiopoietin 2 and Tie-2 interactions may be involved in the preparation of endometrium for implantation, remodelling of the maternal vasculature and trophoblast invasion during the peri-implantation period in this primate species.

Angiogenesis and vascular endothelial growth factor expression in the equine corpus luteum.

Precise pharmacological control of the corpus luteum is important in the manipulation of the oestrous cycle in mares. Angiogenesis plays a key role in the growth and regression of the corpus luteum; therefore, influencing the vasculature of the corpus luteum may offer a novel method for controlling its lifespan. In the present study, changes in angiogenesis and vascular expression of endothelial growth factor (VEGF) were evaluated throughout the luteal phase and after PGF(2alpha)-induced luteolysis. Corpora lutea were collected from mares in the early luteal phase (days 3-4), mid-luteal phase (day 10), early regression (day 14), late regression (day 17), and at 12 and 36 h after administration of PGF(2alpha) on day 10 of the oestrous cycle. Immunohistochemistry was used to localize Von Willebrand factor and Ki67 in endothelial and proliferating cells, respectively. VEGF mRNA and protein were localized by in situ hybridization and immunohistochemistry. The proliferation index of endothelial cells was intense in the early luteal phase. The early and mid-luteal phases were characterized by a dense network of capillaries. The microvasculature started to regress by day 14. After administration of PGF(2alpha), vasodilation was observed after 12 h, but after 36 h, luteal degeneration was accompanied by a significant decrease in vascularity. VEGF mRNA and protein were expressed mainly in the luteal cells during the early and mid-luteal phases and expression declined at early regression (day 14). However, immunostaining for VEGF protein was high in late luteal regression (day 17) and 36 h after PGF(2alpha) administration. These findings indicate a close temporal association between VEGF expression and angiogenesis in the equine corpus luteum during its functional lifespan.

Testicular changes during infantile 'quiescence' in the marmoset and their gonadotrophin dependence: a model for investigating susceptibility of the prepubertal human testis to cancer therapy?

BACKGROUND: Inexplicably, boys treated with some therapies for cancer at age 2-10 years, a time of supposed 'testicular quiescence', are at risk of low sperm counts/infertility in adulthood. Our aims were to use the marmoset as a surrogate for man to establish testicular cell function/activity during 'quiescence' between the neonatal period and puberty, and to test if any cell activity could be suppressed by prior treatment with a GnRH antagonist. METHODS AND RESULTS: Based on immunoexpression studies, functional development of Sertoli cells (SGP-2, androgen receptor) and Leydig cells (3 beta-hydroxysteroid dehydrogenase) was detectable at an age (35 weeks) when the testis is considered to be quiescent, and in advance of the pubertal rise in blood testosterone levels (50-60 weeks). Other changes at 35 weeks were the appearance of focal seminiferous tubule lumens and proliferating germ cells [indicated by immunoexpression of proliferating cell nuclear antigen (PCNA)]. Treatment from 25 to 35 weeks with GnRH antagonist largely (>85%) prevented these changes. However, the PCNA-labelling index of spermatogonia in GnRH antagonist-treated animals did not differ from controls (41.3 versus 43.6%) though total spermatogonia volume per testis was reduced by 41%. Some protein markers (inhibin-alpha, estrogen receptor-beta) showed little change with age or treatment. Beyond 35 weeks, GnRH antagonist-treated animals showed a delay in the pubertal rise in plasma testosterone levels. CONCLUSIONS: These findings reinforce the view that the 'childhood' testis is not quiescent. This may explain the damaging effects of some cancer therapies on subsequent fertility of boys and raises the issue of protective intervention. The present studies suggest that GnRH antagonist-based intervention might be only partially successful. Identification of the factors regulating spermatogonial development in the infant marmoset may aid in the design of such strategies.

Cell death during natural and induced luteal regression in mares.

In mares, little information is available on the type of cell death that occurs during natural and induced luteal regression. Corpora lutea were collected from mares in the early luteal phase, days 3-4 (n = 4); mid-luteal phase, day 10 (n = 5); early regression, day 14 (n = 4); late regression, day 17 (n = 4); and 12 and 36 h (n = 3 per group) after PGF2alpha administration on day 10. Histological and ultrastructural sections were examined and TUNEL was used to detect DNA fragmentation. In early luteal regression, there were more pyknotic luteal cells and extracellular round dense bodies compared with the mid-luteal phase. By late regression, there was a significant decline (P < 0.01) in the number of round dense body clusters and a marked accumulation of lipid. Twelve and 36 h after PGF2alpha administration, changes were similar to those seen in natural regression, but there was also a marked infiltration of neutrophils. Accumulation of lipid was not apparent until 36 h after PGF2alpha administration. Ultrastructural examination revealed rarefaction and distortion of the mitochondrial cristae in most of the luteal cells by the mid-luteal phase. Luteal cells showed shrinkage, accumulation of lipid with foamy appearance, and disruption in both smooth endoplasmic reticulum and mitochondria during natural and induced regression. Some luteal cells showed fragmented or pyknotic chromatin characteristic of apoptosis. Other luteal cells showed crenation of the nuclear membrane and shrinkage of the nucleus, features not characteristic of apoptotic cell death. In late regression, capillaries were obstructed by swollen endothelial cells and round dense bodies. These results show that structural regression may be initiated as early as the mid-luteal phase, and is clearly visible by day 14 in natural regression and 12 h after induced regression. Apoptosis did appear to be involved in luteolysis in the equine corpus luteum, but non-apoptotic changes were also observed in some luteal cells during regression. Accumulation of lipid was a late feature of luteal regression.

Structure of the corpus luteum in the ovulatory polycystic ovary.

BACKGROUND: Women with polycystic ovaries (PCO) have a wide spectrum of presentation from anovulation and amenorrhoea to apparently regular, ovulatory menstrual cycles. We have recently reported a subtle defect in steroidogenic function in the luteal phase in the latter and an increase in the number of degenerating corpora lutea (CL) were observed in ovulatory PCO (ovPCO) during dissection. The possibility was therefore investigated of differences in structure or degeneration in CL formed during ovulatory cycles in women with PCO. METHODS: This study compared the histology of the CL in ovPCO with that in the normal ovary. Corpora lutea were collected from nine normal ovaries (days 1-27 of the cycle) and from 13 women with ovPCO (days 5-38). RESULTS: Variations in the degree of regression, both in relation to onset of menses and between different areas within individual CL, were recorded in both groups. During development and regression no obvious differences were observed between either group apart from an apparent increase in luteal haemorrhage, which was more common and more extensive in CL from PCO. CONCLUSIONS: The findings suggest that possible luteal phase abnormalities of steroid secretion in women with ovulatory PCO are not associated with obvious morphological defects in the CL, however it is possible that the persistence of luteal structures seen in PCO was a consequence of increased luteal haemorrhage.

Development and validation of a new monoclonal antibody to mammalian aromatase.

The biosynthesis of oestrogens from androgens is catalysed by the aromatase complex, an essential component of which is the aromatase cytochrome P450 (P450 arom) protein. Expression of a functional P450 arom is essential for normal fertility in males and females and the sequence of the protein is highly conserved. We have raised a new monoclonal antibody against a conserved peptide and validated it on fixed tissue sections of the rat, common marmoset (Callthrix jacchus) and human. The monoclonal antibody was used successfully for Western analysis and specifically reacted with a 55 kDa protein in microsomal extracts. On sections of ovaries in all three species, expression in follicles was specific to the mural granulosa cells of antral follicles and was present in corpora lutea. In the human and marmoset, staining of luteal cells was markedly heterogeneous and did not appear to vary consistently with the stage of the cycle. The intensity of immunostaining was elevated in corpora lutea from pregnant rats and following human chorionic gonadotropin rescue in the human. In the testis, the highest levels of expression were observed in the Leydig cells within the interstitium. In adult rat and marmoset, and possibly also in the human, some P450 arom was associated with the cytoplasm surrounding elongate spermatids but other germ cells were immunonegative. In conclusion, a new monoclonal antibody specific for P450 arom recognises the protein in rodent, primate and human. Its ability to work on fixed tissue sections will facilitate identification of individual cells expressing P450 arom within complex tissues.

Angiogenesis in the human corpus luteum: simulated early pregnancy by HCG treatment is associated with both angiogenesis and vessel stabilization.

BACKGROUND: This study examined changes in the luteal vasculature throughout the menstrual cycle and during simulated pregnancy with human chorionic gonadotrophin (HCG) in the human. METHODS: Endothelial cell and pericyte area were assessed by quantitative immunocytochemistry for CD34 and alpha-smooth muscle actin respectively, taking into consideration the dynamics of lutein cell hypertrophy and atrophy throughout the cycle and after HCG treatment. Endothelial cell proliferation was detected by Ki-67/CD34 dual staining and a proliferation index was obtained. The molecular regulation of angiogenesis was studied by examining changes in vascular endothelial growth factor (VEGF) immunostaining. RESULTS: The early luteal phase is associated with intense angiogenesis, as indicated by high endothelial cell proliferation, and by the mid-luteal phase a mature vasculature was apparent, as shown by maximal endothelial cell and pericyte areas. During the late luteal phase, decreased endothelial proliferation, endothelial cell and pericyte area indicated vascular regression. HCG treatment induced a second burst of total and endothelial cell proliferation and a concomitant increase in endothelial cell and pericyte areas. VEGF protein was expressed throughout the luteal phase and a significant increase was found after HCG treatment. CONCLUSION: Luteal rescue with HCG is associated with a second wave of angiogenesis and vascular stabilization.