Reproduction

Delbès, Géraldine; Levacher, Christine; Habert, René

doi: 10.1530/rep.1.01231pmid: 17008464

In recent years, evidences have accumulated that exposure to environmental components with estrogenic activity causes reproductive disorders in human populations. Studies conducted over the past 50 years have clearly shown a continual decline in semen quality accompanied by an increase in male reproductive disorders during this period in industrial countries. As healthy gametes are a prerequisite for healthy children, such disorders are a significant problem not only for the current society, but also for future generations. These male reproductive disorders have been attributed to xenobiotics, and particularly to xenoestrogens, which have steadily increased in diversity and concentration in the environment and food. Epidemiological, clinical, and experimental studies have suggested that excessive exposure to estrogens and xenoestrogens during fetal and neonatal development may induce testicular developmental disorders, leading to alterations in the adult male fertility. Recently, we have clearly demonstrated that fetal and neonatal testes are very sensitive to estrogens, as the inactivation of estrogen receptor α increases steroidogenesis and the inactivation of estrogen receptor β enhances development of the germ cell lineage in the male.

Robinson, Jane

doi: 10.1530/rep.1.00064pmid: 17008465

It has been clear for several decades that the areas of the brain that control reproductive function are sexually dimorphic and that the ‘programming actions’ of the male gonadal steroids are responsible for sex-specific release of the gonadotrophins from the pituitary gland. The administration of exogenous steroids to fetal/neonatal animals has pinpointed windows of time in an animals’ development when the reproductive neuroendocrine axis is responsive to the organisational influences of androgens. These ‘critical’ periods for sexual differentiation of the brain are trait- and species-specific. The neural network regulating the activity of the gonadotrophin releasing hormone (GnRH) neurones is vital to the control of reproductive function. It appears that early exposure to androgens does not influence the migratory pathway of the GnRH neurone from the olfactory placode or the size of the population of neurones that colonise the postnatal hypothalamus. However, androgens do influence the number and the nature of connections that these neurones make with other neural phenotypes. Gonadal steroid hormones play key roles in the regulation of GnRH release acting largely via steroid-sensitive intermediary neurones that impinge on the GnRH cells. Certain populations of hormonally responsive neurones have been identified that are sexually dimorphic and project from hypothalamic areas known to be involved in the regulation of GnRH release. These neurones are excellent candidates for the programming actions of male hormones in the reproductive neuroendocrine axis of the developing female.

Ikeda, S; Saeki, K; Imai, H; Yamada, M

doi: 10.1530/rep.1.01066pmid: 17008466

We previously reported that when midkine (MK), a heparin-binding growth differentiation factor was used in in vitro maturation (IVM) culture of bovine cumulus-enclosed oocytes (CEOs), their developmental competence to the blastocyst stage after in vitro fertilization (IVF) was enhanced and the effect of MK might be mediated by its action upon mural granulosa cells and cumulus cells that closely surround the oocyte. In the present study, when denuded oocytes (DOs) were matured in IVM medium with or without MK (200 ng/ml) in the presence or absence of isolated cumulus cell masses and subjected to IVF, the enhancing effects of MK on the developmental competence of DOs to the blastocyst stage after IVF were exerted only in the presence of cumulus cells. In addition, we prepared the conditioned media of granulosa cells cultured with or without 200 ng MK/ml (CMMK+ or CMMK− respectively) and examined their effects on the IVM of DOs in terms of their developmental competence to the blastocyst stage after IVF. The supplementation of CMMK+ into IVM medium at 40% (v/v) significantly enhanced the blastocyst development compared with the no additive control and the CMMK− supplemented groups. Furthermore, the effects of MK during IVM of bovine CEOs on the cumulus cell apoptosis were investigated. CEOs were cultured up to 24 h in IVM medium without (control) or with 200 ng MK/ml. The genomic DNA was extracted from CEOs at 0, 6, 12, 18 and 24 h of IVM and subjected to ligation-mediated PCR (LM-PCR) to detect the apoptotic internucleosomal DNA fragmentation. DNA fragmentation was scarcely detected at the start of IVM, whereas it increased time-dependently as the IVM culture progressed. The degree of the fragmentation was significantly lower in the MK-treatment group compared with the control group at 18 and 24 h of IVM. The apoptosis-suppressing effect of MK on cumulus cells was further confirmed in situ by using TUNEL on CEOs. In conclusion, data from the present study further confirmed that MK enhances the developmental competence of bovine oocytes via cumulus and granulosa cells. It was also demonstrated that MK suppresses the apoptosis that occurs in cumulus cells during the period of IVM of bovine CEOs. The putative soluble factor(s) from cumulus cells was suggested from the experiment using CMMK+ . MK may promote the production of such factors in part by its anti-apoptotic effects on cumulus cells.

Somfai, Tamás; Ozawa, Manabu; Noguchi, Junko; Kaneko, Hiroyuki; Ohnuma, Katsuhiko; Karja, Ni Wayan Kurniani; Fahrudin, Mokhamad; Maedomari, Naoki; Dinnyés, András; Nagai, Takashi; Kikuchi, Kazuhiro

doi: 10.1530/rep.1.01216

Gaytán, M; Bellido, C; Morales, C; Sánchez-Criado, J E; Gaytán, F

doi: 10.1530/rep.1.01236pmid: 17008468

Treatment with non-steroidal anti-inflammatory drugs, either non-selective or selective cyclooxygenase-2 (COX-2) inhibitors, consistently impairs ovulation, indicating the essential role of COX-2/prostaglandins in the ovulatory process. Indomethacin, a potent inhibitor of both COX-1 and COX-2, induced several ovulatory alterations, consisting of a decrease in the number of oocytes effectively ovulated, trapping of oocytes inside the luteinized follicle, as well as abnormal follicle rupture at the basolateral sides, with release of the oocyte and follicular fluid to the interstitium. Yet, the precise role of prostaglandins in ovulation and whether some of the ovulatory defects induced by indomethacin are due to interference with additional components of the ovulatory cascade, beyond prostaglandin synthesis, are not completely understood. We have used gonadotrophin-primed immature rats to analyse whether, compared to indomethacin, selective inhibition of COX-2, with or without concomitant inhibition of COX-1, or selective inhibition of the lipooxygenase (LOX) pathway, induce similar ovulatory alterations. Immature rats (27 days of age) were injected PMSG (10 IU), and 48 h later hCG (10 IU) subcutaneously, and different anti-inflammatory drugs. Animals were killed at 21 h after hCG injection. Rats treated with the selective COX-2 inhibitor NS398 (10 mg/kg body weight, (bw)) showed alterations in follicle rupture as those treated with indomethacin (0.5 mg/rat), albeit affecting a lower number of follicles, irrespective of the concomitant inhibition of COX-1 with the selective inhibitor SC560 (10 mg/kg bw). Rats treated with the LOX inhibitor NDGA (300 mg/kg bw) did not show ovulatory alterations. These data indicate that the characteristic alterations of follicle rupture induced by indomethacin, are also induced by selective COX-2 inhibitors, strengthening the contention that prostaglandins play a crucial role in the spatial targeting of follicle rupture at the apex.

Grazul-Bilska, Anna T; Navanukraw, Chainarong; Johnson, Mary Lynn; Arnold, Daniel A; Reynolds, Lawrence P; Redmer, Dale A

doi: 10.1530/REP-06-0009pmid: 17008469

This study was conducted to evaluate the expression of endothelial nitric oxide synthase (eNOS) in ovarian follicles and corporalutea (CL) throughout the estrous cycle in sheep. Three experiments were conducted to (1) immunolocalize eNOS protein, (2)determine expression of mRNA for eNOS and its receptor guanylate cyclase 1 soluble β3 (GUCY1B3), and (3) co-localize eNOSand vascular endothelial growth factor (VEGF) proteins in the follicles and/or CL throughout the estrous cycle. In experiment1, ovaries were collected from ewes treated with FSH, to induce follicular growth or atresia. In experiment 2, ovaries werecollected from ewes treated with FSH and hCG to induce follicular growth and ovulation. In experiment 3, ovaries were collectedfrom superovulated ewes to generate multiple CL on days 2, 4, 10, and 15 of the estrous cycle. In experiments 1 and 2, theexpression of eNOS protein was detected in the blood vessels of the theca externa and interna of healthy ovarian follicles.However, in early and advanced atretic follicles, eNOS protein expression was absent or reduced. During the immediate postovulatoryperiod, eNOS protein expression was detected in thecal-derived cells that appeared to be invading the granulosa layer. Expressionof eNOS mRNA tended to increase in granulosa cells at 12 and 24 h, and in theca cells 48 h after hCG injection. In experiment3, eNOS protein was located in the blood vessels of the CL during the estrous cycle. Dual localization of eNOS and VEGF proteinsin the CL demonstrated that both were found in the blood vessels.

Grazul-Bilska, Anna T; Navanukraw, Chainarong; Johnson, Mary Lynn; Arnold, Daniel A; Reynolds, Lawrence P; Redmer, Dale A

doi: 10.1530/rep-06-0009pmid: 17008469

This study was conducted to evaluate the expression of endothelial nitric oxide synthase (eNOS) in ovarian follicles and corpora lutea (CL) throughout the estrous cycle in sheep. Three experiments were conducted to (1) immunolocalize eNOS protein, (2) determine expression of mRNA for eNOS and its receptor guanylate cyclase 1 soluble β3 (GUCY1B3), and (3) co-localize eNOS and vascular endothelial growth factor (VEGF) proteins in the follicles and/or CL throughout the estrous cycle. In experiment 1, ovaries were collected from ewes treated with FSH, to induce follicular growth or atresia. In experiment 2, ovaries were collected from ewes treated with FSH and hCG to induce follicular growth and ovulation. In experiment 3, ovaries were collected from superovulated ewes to generate multiple CL on days 2, 4, 10, and 15 of the estrous cycle. In experiments 1 and 2, the expression of eNOS protein was detected in the blood vessels of the theca externa and interna of healthy ovarian follicles. However, in early and advanced atretic follicles, eNOS protein expression was absent or reduced. During the immediate postovulatory period, eNOS protein expression was detected in thecal-derived cells that appeared to be invading the granulosa layer. Expression of eNOS mRNA tended to increase in granulosa cells at 12 and 24 h, and in theca cells 48 h after hCG injection. In experiment 3, eNOS protein was located in the blood vessels of the CL during the estrous cycle. Dual localization of eNOS and VEGF proteins in the CL demonstrated that both were found in the blood vessels.

Fraser, Hamish M; Wilson, Helen; Wulff, Christine; Rudge, John S; Wiegand, Stanley J

doi: 10.1530/rep.1.01064pmid: 17008470

The intense angiogenesis characteristic of early corpus luteum development is dependent upon vascular endothelial growth factor (VEGF) as inhibitors of VEGF administered at the peri-ovulatory period suppress endothelial cell proliferation and progesterone secretion. We now report that administration of VEGF Trap, a soluble decoy receptor-based inhibitor, at the mid- or the late luteal phase in the marmoset results in a rapid decline in plasma progesterone. Since vascularisation of the corpus luteum is largely complete by the mid-luteal phase, it suggested that this functional luteolysis involved mechanisms other than inhibition of angiogenesis. A second experiment investigated the role of VEGF in maintaining the integrity of the luteal vasculature and hormone-producing cells. VEGF Trap was administered to marmosets in the mid-luteal phase and ovaries were obtained 1, 2, 4 or 8 days later for localisation of activated caspase-3 staining in the corpus luteum and compared with those obtained 2, 4 and 8 days after administration of control protein. The number of cells with activated caspase-3 staining was significantly increased after administration of VEGF Trap. Dual staining of activated caspase-3 with the endothelial cell marker CD31 showed that at 1 day post-treatment, more than 90% caspase-3-stained cells were vascular endothelium, prior to detection of an increasing incidence in death of hormone-producing cells on days 2 and 4. Staining with CD31 showed that the endothelial cell area was decreased after treatment. By 8 days after treatment, corpora lutea had regressed to varying degrees, while all control corpora lutea remained healthy. These results show that VEGF inhibition in the mid- or the late luteal phase induces functional luteolysis in the marmoset that is associated with premature and selective death of endothelial cells.

Lin, S-Y; Morrison, J R; Matzuk, M M; de Kretser, D M

doi: 10.1530/rep.1.01172pmid: 17008471

It has been proposed that follistatin can modulate the actions of activins and/or other members of the transforming growth factor-β superfamily of proteins on testicular function, since mice overexpressing follistatin showed spermatogenic disruption. However, since mice with targeted disruption of the follistatin gene die soon after birth, it is not feasible to determine the effect of the absence of follistatin on testicular function using this model. To further understand the role of follistatin on the development and maintenance of spermatogenesis, fetal testes, collected by Caesarean section at day 18 of gestation from follistatin null mice, were transplanted to the external ear of castrated recombination activating gene 1 immunocompromised male mice. The testicular grafts were then analysed 7–8 weeks after transplantation and showed that full spermatogenesis developed in both the testes of wild-type and follistatin null mice. This study indicates that, if follistatin is required to modulate spermatogenic development, it is not supplied by local testicular production but by circulating follistatin from the host mouse.

Kahiri, Caroline N; Khalil, M Wahid; Tekpetey, Francis; Kidder, Gerald M

doi: 10.1530/rep.1.01234pmid: 17008472

Connexin43 (Cx43) is the most abundantly expressed member of the connexin (gap junction protein) family and the only one so far identified in mouse Leydig cell gap junctions. Mice lacking Cx43 were used to investigate its role in testicular androgen production and regulation. Testes from term fetuses were grafted under the kidney capsules of castrated adult males. After 3 weeks, serum from host mice was analyzed for androgens. In order to test their response to stimulation, the grafted testes were incubated in vitro with varying concentrations of LH and their androgen end products analyzed. Incubation with radiolabeled progesterone was followed by high performance liquid chromatography to quantify the androgen-intermediate metabolites. Radiolabeled testosterone in the presence of NADPH was used to determine the activity of testosterone-metabolizing enzymes 17β-hydroxysteroid dehydrogenase (17βHSD), 5α-reductase (5αR), and 3α-hydroxysteroid dehydrogenase (3α HSD). Serum androgen levels did not differ between hosts carrying wild-type versus null mutant grafts although Cx43-deficient testes had more 17βHSD and 5αR activity than wild-type controls. Furthermore, the genotype of grafted testes did not influence LH-stimulated androgen production in vitro. These results indicate that the steroidogenic function of Leydig cells is not compromised by the absence of Cx43, perhaps because other gap junction proteins are present. Dye transfer experiments demonstrated that Cx43-deficient Leydig cells retain intercellular coupling, indicating that Cx43 is not the only protein contributing to their gap junctions. Thus, despite their prominence in Leydig cells, Cx43 gap junctions are not essential for androgen production.