Reproduction

Zheng, Xiaofeng; Price, Christopher A; Tremblay, Yves; Lussier, Jacques G; Carrière, Paul D

doi: 10.1530/rep-07-0316pmid: 18635743

Survival and inhibitory factors regulate steroidogenesis and determine the fate of developing follicles. The objective of this study was to determine the role of transforming growth factor-β1 (TGFB1) in the regulation of estradiol-17β (E2) and progesterone (P4) secretion in FSH-stimulated bovine granulosa cells. Granulosa cells were obtained from 2 to 5 mm follicles and cultured in serum-free medium. FSH dose (1 and 10 ng/ml for 6 days) and time in culture (2, 4, and 6 days with 1 ng/ml FSH) increased E2 secretion and mRNA expression of E2-related enzymes cytochrome P450 aromatase (CYP19A1) and 17β-hydroxysteroid dehydrogenase type 1 (HSD17B1), but not HSD17B7. TGFB1 in the presence of FSH (1 ng/ml) inhibited E2 secretion, and decreased mRNA expression of FSH receptor (FSHR), CYP19A1, and HSD17B1, but not HSD17B7. FSH dose did not affect P4 secretion and mRNA expression of 3β-hydroxysteroid dehydrogenase (HSD3B) and α-glutathione S-transferase (GSTA), but inhibited the amount of steroidogenic acute regulatory protein (STAR) mRNA. Conversely, P4 and mRNA expression of STAR, cytochrome P450 side-chain cleavage (CYP11A1), HSD3B, and GSTA increased with time in culture. TGFB1 inhibited P4 secretion and decreased mRNA expression of STAR, CYP11A1, HSD3B, and GSTA. TGFB1 modified the formation of granulosa cell clumps and reduced total cell protein. Finally, TGFB1 decreased conversion of androgens to E2, but did not decrease the conversion of estrone (E1) to E2 and pregnenolone to P4. Overall, these results indicate that TGFB1 counteracts stimulation of E2 and P4 synthesis in granulosa cells by inhibiting key enzymes involved in the conversion of androgens to E2 and cholesterol to P4 without shutting down HSD17B reducing activity and HSD3B activity.

Gassei, Kathrin; Ehmcke, Jens; Schlatt, Stefan

doi: 10.1530/rep-08-0241pmid: 18660385

The first morphological sign of testicular differentiation is the formation of testis cords. Prior to cord formation, newly specified Sertoli cells establish adhesive junctions, and condensation of somatic cells along the surface epithelium of the genital ridge occurs. Here, we show that Sertoli cell aggregation is necessary for subsequent testis cord formation, and that neurotrophic tyrosine kinase receptors (NTRKs) regulate this process. In a three-dimensional cell culture assay, immature rat Sertoli cells aggregate to form large spherical aggregates (81.36±7.34 μm in diameter) in a highly organized, hexagonal arrangement (376.95±21.93 μm average distance between spherical aggregates). Exposure to NTRK inhibitors K252a and AG879 significantly disrupted Sertoli cell aggregation in a dose-dependent manner. Sertoli cells were prevented from establishing cell–cell contacts and from forming spherical aggregates. In vitro-derived spherical aggregates were xenografted into immunodeficient nude mice to investigate their developmental potential. In controls, seminiferous tubule-like structures showing polarized single-layered Sertoli cell epithelia, basement membranes, peritubular myoid cells surrounding the tubules, and lumen were observed in histological sections. By contrast, grafts from treatment groups were devoid of tubules and only few single Sertoli cells were present in xenografts after 4 weeks. Furthermore, the grafts were significantly smaller when Sertoli cell aggregation was disrupted by K252a in vitro (20.87 vs 6.63 mg; P<0.05). We conclude from these results that NTRK-regulated Sertoli–Sertoli cell contact is essential to the period of extensive growth and remodeling that occurs during testicular tubulogenesis, and our data indicate its potential function in fetal and prepubertal testis differentiation.

Kumar, Priyadarsini; Meizel, Stanley

doi: 10.1530/REP-08-0223pmid: 18621921

The human sperm surface glycine receptor (GLR) plays a role in an important fertilization event, the sperm acrosome reaction.Here, by western blot analysis, we report the presence of GLRA1, GLRA2, GLRA3, and GLRB subunits in human sperm. Immunolocalizationstudies showed that the GLRA1 and GLRA2 subunits are present in the equatorial region, the GLRA3 subunit in the flagellarprincipal piece, and the GLRB subunit in the acrosomal region of sperm. This first demonstration of isoforms of the spermGLRA subunit and of a differential spatial distribution of the α and β subunits on the surface of mammalian sperm suggeststhe possibility that human sperm GLRs have more than one function.

Chaturapanich, G; Chaiyakul, S; Verawatnapakul, V; Pholpramool, C

doi: 10.1530/REP-08-0069pmid: 18614624

Krachaidum (KD, Kaempferia parviflora Wall. Ex. Baker), a native plant of Southeast Asia, is traditionally used to enhance male sexual function. However, onlyfew scientific data in support of this anecdote have been reported. The present study investigated the effects of feedingthree different extracts of KD (alcohol, hexane, and water extracts) for 3–5 weeks on the reproductive organs, the aphrodisiacactivity, fertility, sperm motility, and blood flow to the testis of male rats. Sexual performances (mount latency, mountfrequency, ejaculatory latency, post-ejaculatory latency) and sperm motility were assessed by a video camera and computer-assistedsperm analysis respectively, while blood flow to the testis was measured by a directional pulsed Doppler flowmeter. The resultsshowed that all extracts of KD had virtually no effect on the reproductive organ weights even after 5 weeks. However, administrationof the alcohol extract at a dose of 70 mg/kg body weight (BW)/day for 4 weeks significantly decreased mount and ejaculatorylatencies when compared with the control. By contrast, hexane and water extracts had no influence on any sexual behavior parameters.All types of extracts of KD had no effect on fertility or sperm motility. On the other hand, alcohol extract produced a significantincrease in blood flow to the testis without affecting the heart rate and mean arterial blood pressure. In a separate study,an acute effect of alcohol extract of KD on blood flow to the testis was investigated. Intravenous injection of KD at dosesof 10, 20, and 40 mg/kg BW caused dose-dependent increases in blood flow to the testis. The results indicate that alcoholextract of KD had an aphrodisiac activity probably via a marked increase in blood flow to the testis.

Ohinata, Yasuhide; Sano, Mitsue; Shigeta, Mayo; Yamanaka, Kaori; Saitou, Mitinori

doi: 10.1530/REP-08-0053pmid: 18583473

The ability to monitor the development of a given cell lineage in a non-invasive manner by fluorescent markers both in vivo and in vitro provides a great advantage for the analysis of the lineage of interest. To date, a number of transgenic or knock-in mousestrains, in which developing germ cells are marked with fluorescent reporters, have been generated. We here describe a noveldouble transgenic reporter mouse strain that expresses membrane-targeted Venus (mVenus), a brighter variant of yellow fluorescentprotein (YFP), under the control of Prdm1 (Blimp1) regulatory elements and enhanced cyan fluorescent protein (ECFP) under the control of Dppa3 (Stella/Pgc7). The double transgenic strain unambiguously marked Prdm1 expression in the lineage-restricted precursors of primordial germ cells (PGCs) in the proximal epiblast at embryonic day(E) 6.25 and specifically illuminated Prdm1- and Dppa3-positive migrating PGCs after E8.5. The double transgenic reporter also precisely recapitulated dynamic embryonic expressionof Prdm1 outside the germ cell lineage. Moreover, we derived ES cells that bore both transgenes. These cells made a robust contributionboth to the germ and somatic cell lineages in chimeras with accurate Prdm1-mVenus and Dppa3-ECFP expression. The transgenic strain and the ES cells will serve as valuable experimental materials not only for analyzingthe origin and properties of the germ cell lineage in vivo, but also for establishing a culture system to efficiently induce proper germ cells with temporally coordinated Prdm1 and Dppa3 expression in vitro.

Gassei, Kathrin; Ehmcke, Jens; Schlatt, Stefan

doi: 10.1530/REP-08-0241pmid: 18660385

The first morphological sign of testicular differentiation is the formation of testis cords. Prior to cord formation, newlyspecified Sertoli cells establish adhesive junctions, and condensation of somatic cells along the surface epithelium of thegenital ridge occurs. Here, we show that Sertoli cell aggregation is necessary for subsequent testis cord formation, and thatneurotrophic tyrosine kinase receptors (NTRKs) regulate this process. In a three-dimensional cell culture assay, immaturerat Sertoli cells aggregate to form large spherical aggregates (81.36±7.34 μm in diameter) in a highly organized, hexagonalarrangement (376.95±21.93 μm average distance between spherical aggregates). Exposure to NTRK inhibitors K252a and AG879 significantlydisrupted Sertoli cell aggregation in a dose-dependent manner. Sertoli cells were prevented from establishing cell–cell contactsand from forming spherical aggregates. In vitro-derived spherical aggregates were xenografted into immunodeficient nude mice to investigate their developmental potential.In controls, seminiferous tubule-like structures showing polarized single-layered Sertoli cell epithelia, basement membranes,peritubular myoid cells surrounding the tubules, and lumen were observed in histological sections. By contrast, grafts fromtreatment groups were devoid of tubules and only few single Sertoli cells were present in xenografts after 4 weeks. Furthermore,the grafts were significantly smaller when Sertoli cell aggregation was disrupted by K252a in vitro (20.87 vs 6.63 mg; P<0.05). We conclude from these results that NTRK-regulated Sertoli–Sertoli cell contact is essential to the period of extensivegrowth and remodeling that occurs during testicular tubulogenesis, and our data indicate its potential function in fetal andprepubertal testis differentiation.

Oliveira, Lilian J; Hansen, P J

doi: 10.1530/REP-08-0218pmid: 18635742

The presence of conceptus alloantigens necessitates changes in maternal immune function. Here, we used the cow to evaluatewhether species with epitheliochorial placentation have changes in specific leukocyte populations during pregnancy similarto those reported in species with hemotropic placentae. At days 33–34 of pregnancy, there was no effect of pregnancy statuson the number of cells positive for CD8, CD4, γδT cell receptor, or the monocyte marker CD68 in the peripheral blood mononuclearcell (PBMC) population. There was, however, an increase in the proportion of CD4+ cells that were positive for CD25. There was no effect of status on the proportion of PBMCs that were CD8+ when comparing preparturient cows to nonpregnant cows. However, preparturient cows had an increased percentage of PBMCs thatwere γδT cells and CD4+CD25+ and a tendency for a lower percentage that were CD68+ cells. Using immunolocalization with anti-CD68, it was found that pregnant cows had increased numbers of CD68+ cells in the endometrial stroma as early as days 54–100 of gestation; this increase persisted through the last time examined(day 240 of gestation). Cells positive for CD68 were also positive for another macrophage/monocyte marker, CD14. In conclusion,pregnancy in the cow is associated with changes in peripheral and endometrial leukocyte numbers, which are similar to patternsobserved in other species.

Kumar, Priyadarsini; Meizel, Stanley

doi: 10.1530/rep-08-0223pmid: 18621921

The human sperm surface glycine receptor (GLR) plays a role in an important fertilization event, the sperm acrosome reaction. Here, by western blot analysis, we report the presence of GLRA1, GLRA2, GLRA3, and GLRB subunits in human sperm. Immunolocalization studies showed that the GLRA1 and GLRA2 subunits are present in the equatorial region, the GLRA3 subunit in the flagellar principal piece, and the GLRB subunit in the acrosomal region of sperm. This first demonstration of isoforms of the sperm GLRA subunit and of a differential spatial distribution of the α and β subunits on the surface of mammalian sperm suggests the possibility that human sperm GLRs have more than one function.

Ethier-Chiasson, M; Forest, J-C; Giguère, Y; Masse, A; Marseille-Tremblay, C; Lévy, E; Lafond, J

doi: 10.1530/rep-08-0082pmid: 18599643

The lectin-like oxidized low-density lipoprotein (LDL) receptor-1 (OLR1) is a newly described receptor for oxidatively modified LDL. The human pregnancy is associated with hyperlipidemia and oxidative stress. It has been reported that modification in maternal lipid profile can induce disturbance during pregnancy. In this study, we have evaluated the expression protein level of OLR1 in human term placenta of women having plasma cholesterol level lower to 7 mM or higher to 8 mM and women of gestational diabetes mellitus (GDM) by western blot analysis. The present study demonstrates that the maternal lipid profile is associated with placental protein expression of OLR1. A significant increase in the protein expression of OLR1 was observed in placenta of women with elevated plasmatic total cholesterol level (>8 mM). In addition, the placental protein expression of OLR1 is increased in mothers having the highest pre-pregnancy body mass index (BMI) and low (<7 mM) plasmatic total cholesterol level at term. Interestingly, the placental protein expression of OLR1 is increased in the presence of GDM pregnancies compared with normal lipids level pregnancies, without the modification of mRNA expression. In conclusion, placental OLR1 protein expression is associated with maternal lipid profile, pre-pregnancy BMI, and pathology of GDM.

Hernández-López, Leonor; Cerda-Molina, Ana Lilia; Páez-Ponce, Denisse L; Mondragón-Ceballos, Ricardo

doi: 10.1530/rep-08-0135pmid: 18647842

In addition to gametes, mammalian internal fertilisation has required the evolution of assorted anatomical, physiological and biochemical devices to deal with intra- and inter-sexual conflict such as sperm competition and female cryptic choice respectively. The seminal coagulum of primates and other mammals is viewed as one of such devices. Among primates, the seminal coagulum characteristically occurs in multi-male and multi-female species, leading us to suppose that it intervenes in sperm competition. However, it can also provide cues to the female reproductive tract about male desired or undesired traits, and therefore deter or favour sperm survival and migration. The present work investigates whether the seminal coagulum of the black-handed spider monkey enhances sperm fertilisation chances by improving the female reproductive tract conditions, and if the female reproductive tract is ‘blind’ to semen or behaves selectively towards ejaculates of different males. A series of artificial inseminations were done in five females, using the ejaculates of three different males, one at a time, and measuring the presence of distinct types of sperm inside the uteri at 10, 30 and 60 min following the insemination. The presence of coagulum, menstrual phase, and male and female identity only affected fast, straight-moving sperm, with larger amounts of fast sperm appearing inside the uteri when ejaculates had seminal coagulum, as well as when in the periovulatory phase. There was great intra-uterine fast-sperm variation regarding which male's semen inseminated which female. The results provide evidence to account for sexual conflict in the spider monkey as well as a methodological approach to this kind of study.