Almuth Einspanier

Activation of Neural Pathways Associated with Sexual Arousal in Non-Human Primates

To evaluate brain activity associated with sexual arousal, fully conscious male marmoset monkeys were imaged during presentation of odors that naturally elicit high levels of sexual activity and sexual motivation.

Relaxin peptides are new global players.

Relaxin (RLX) has come of age. From being one of the earliest hormones described with a very specific function in parturition, recent research has now shown that it is involved in a variety of roles, from endometrial differentiation during embryo implantation, to being a response factor in infarct and wound situations. It ameliorates fibrosis, and might also be involved in tumour growth and progression. And it is not alone: two other closely related peptide hormones have recently been identified, one specific for the brain, the other with roles in testicular descent and ovarian apoptosis. Finally, the recent cloning of the RLX receptors now provides the basis for a new molecular pharmacology for these peptide hormones, and preliminary studies suggest that their signal transduction is both interesting and unusual.

Expression of vascular endothelial growth factor (VEGF) and its corresponding receptors (flt-1 and flk-1) in the bovine oviduct.

Vascular endothelial growth factor (VEGF) functions as a potent angiogenic protein as well as in regulating permeability. Reverse transcription‐polymerase chain reaction (RT‐PCR) and ribonuclease protection assay (RPA) were used to show that the bovine oviduct expresses VEGF and its two receptors flk‐1 and flt‐1. Expression of VEGF was relatively stable during the estrous cycle. In contrast, both receptor transcripts showed cycle‐dependent variations with significantly increased flt‐1 mRNA amounts before ovulation. Immunohistochemical studies localized VEGF mainly on the epithelial surface of oviducts. Protein concentrations of VEGF in oviductal flushings were significantly higher (mean ± SEM: 2.8 ± 0.8 ng/ml) during the pre‐ovulatory phase when compared with the other estrous cycle stages (1.0 ± 0.25 ng/ml).

Bioactivity of recombinant prorelaxin from the marmoset monkey

The hormone relaxin (RLX) is generally present in the serum of humans and primates as a heterodimer, though some unprocessed prohormone may also be present. In order to test whether this proRLX is biologically relevant for human or primate physiology, recombinant marmoset monkey proRLX was synthesized in a baculovirus-infected cell system and tested in different bioassays. Marmoset proRLX is >70% identical to human H2 proRLX, especially in the so-called receptor-binding region of the B-peptide. The bioassay systems used were (a) cAMP production by human endometrial stromal cells and (b) cAMP production by the human monocyte cell line THP-1. In both bioassay systems recombinant proRLX showed comparable EC(50) values to pure porcine heterodimeric relaxin (porcine relaxin, 1.5-2.0 nM; marmoset prorelaxin 4.0-5.0 nM). Additionally, recombinant marmoset prorelaxin was shown to stimulate steroidogenesis in primary cultures of marmoset ovarian theca cells, though with a lower apparent activity than porcine relaxin. It thus appears that precursor processing of human or primate relaxin is not an essential prerequisite for the acquisition of bioactivity, as it is for the closely related hormone insulin, and that circulating prorelaxin is physiologically relevant.

Gender and gonadal status differences in zona reticularis expression in marmoset monkey adrenals : Cytochrome b5 localization with respect to cytochrome P450 17,20-lyase activity

Neonatal marmosets express an adrenal fetal zone comparable to humans. While adult males fail to express a functional ZR, with barely detectable blood DHEA levels, females produce higher levels of DHEA than males in adulthood. We investigated the presence of a putative functional ZR in adult female marmosets. In contrast to males, immunohistochemical analysis showed the ZR marker cytochrome b5 was elevated in the innermost zone in cycling females (compared to testis-intact males), further elevated in the adrenals from anovulatory females, and substantially elevated and continuous in ovariectomized females. As a functional test in vivo, following overnight dexamethasone treatment, cycling and anovulatory females showed higher levels of DHEA relative to males, but DHEA failed to increase in response to ACTH. In direct contrast, while ovariectomized females exhibited lower initial DHEA levels, clear increases were detectable after ACTH administration (p<0.05), suggesting an adrenal origin. The apparent differences in cytochrome b5 expression between groups were also further verified by Western blotting of adrenal microsomes, and compared to 17,20-lyase activity; the two parameters were positively correlated (p<0.01) across multiple treatment groups. We conclude that the cycling female marmoset expresses a rudimentary ZR with at least a capacity for DHEA production that becomes significantly ACTH-responsive after anovulation. Expression of cytochrome b5 in this region may be directly or indirectly controlled by gonadal function, and is, at least in part, a critical determinant in the development of an adrenal ZR that is more defined and significantly ACTH-responsive.

Mechanisms of estradiol inactivation in primate endometrium.

In uterine endometrium, the level of estradiol is controlled by oxidative 17beta-hydroxysteroid dehydrogenase (17HSD) activity which converts the bioactive hormone to the less active compound estrone. At least three different types of 17HSD (types 2, 4 and 8) use estradiol as their preferred substrate and may contribute to the overall rate of estradiol-inactivation in the uterus. In this study the marmoset monkey (Callithrix jacchus) was used for the investigation of the particular contribution of each type of 17HSD. Northern Blots revealed essentially the same tissue distribution as in the human. Likewise, uterine 17HSD enzyme activity increases in the secretory phase of the reproductive cycle, in parallel to the rise in circulating progesterone levels. Northern analysis of uteri from defined time points of the reproductive cycle showed that only the level of 17HSD2 expression is strongly upregulated in the secretory phase, whereas 17HSD4 and 17HSD8 seem to be expressed constitutively.

Characterization of 17β-Hydroxysteroid Dehydrogenase Type 7 in Reproductive Tissues of the Marmoset Monkey

Abstract In contrast to the known rodent enzymes, the physiological significance of 17β-hydroxysteroid dehydrogenase type 7 (17HSD7) and its presumed function in reproductive biology is not well understood in primates. As a first step, we recently cloned the complete coding regions of human and marmoset monkey (Callithrix jacchus) 17HSD7 (cj17HSD7). In the present work the complete cDNA of marmoset 17HSD1 (cj17HSD1), including the proximal promoter region, and a partial sequence of marmoset aromatase (cjARO) were sequenced in order to compare the expression of these estradiol synthesizing enzymes with that of 17HSD7 in a primate model and to identify tissues where 17HSD7 might participate in the pathway of estradiol synthesis. The gene structures of cj17HSD1 and cj17HSD7 were determined and proved to be very similar to the human orthologues. Northern hybridization showed that cjARO mRNA seems to be coexpressed preferably with cj17HSD1 in placenta, whereas in other tissues it is expressed in parallel only with cj17HSD7. Especially in corpora lutea, the cj17HSD7 transcript is detectable throughout the luteal phase of the ovarian cycle and increases during pregnancy, in parallel with the transcript of aromatase. Results were confirmed by immunoblots and immunohistochemistry using new polyclonal antisera directed against cj17HSD7 and cjARO protein. The enzymatic conversion of estrone to estradiol was assessed in marmoset corpora lutea. The pattern of coexpression with aromatase supports the hypothesis that luteal 17HSD7 complements placental 17HSD1, ensuring continued estradiol synthesis throughout pregnancy in primates.

Relaxin is an important factor for uterine differentiation and implantation in the marmoset monkey

Relaxin is known to be associated with changes in the uterus and cervix in relation to birth, particularly in terms of a softening and widening of the birth canal and at least in some species a suppression of spontaneous contractions [1,2]. Such findings are generally observed in animal models, like the rat and pig, which show a marked relaxin physiology with maximum relaxin levels during the last trimester of pregnancy. For primates, however, this classic role of relaxin is open to question.

Expression of the estradiol-synthesizing 17ß-hydroxysteroid dehydrogenases type 1 and type 7 in the nonhuman primate Callithrix jacchus

The common marmoset monkey (Callithrix jacchus) was used as a primate model for the study of the expression of the estradiol synthesizing enzymes 17beta-hydroxysteroid dehydrogenase type 1 and type 7 (17HSD1 and 17HSD7). The tissue-specific expression of 17HSD1 and 17HSD7 mRNA in Callithrix jacchus and human as shown by Northern Blot analysis revealed strong similarities between the two species. After cloning of the marmoset-specific coding cDNA sequence of 17HSD7 a similarity of 95% to the known human sequences was found. To elucidate the physiological function of 17HSD7 which is thought to be different to that of the well-known 17HSD1, the regulation of 17HSD7 expression in the corpus luteum was investigated. It was shown to be upregulated during the luteal phase of the reproductive cycle and during early pregnancy, when the primate corpus luteum is most active in estradiol synthesis, whereas 17HSD1 was not detectable in this tissue at any time.

The Common Marmoset Monkey as a Model for Implantation and Early Pregnancy Research

This chapter describes methods used to investigate implantation in the common marmoset monkey, Callithrix jacchus. A reverse-transcriptase polymerase chain reaction-strategy with which to detect transcripts for steroid receptors and enzymes involved in estradiol biosynthesis is described, and an immunohistochemistry approach for detecting proteins within the implantation site is presented.