The hamster cheek pouch: an immunologically privileged site suitable to the study of granulomatous infections

A bolsa jugal do hamster (BJH) é uma invaginação da mucosa oral, caracterizada histologicamente como semelhante a pele. Nesse estudo nós descrevemos algumas de suas características anatômicas, histológicas e embriológicas e comentamos sobre sua propriedade como local imunologicamente privilegiado, considerando a ausência de drenagem linfática e o reduzido número de células de Langerhans. Apresentamos também os resultados obtidos quando da inoculação de micobacterias (BCG, Mycobacterium tuberculosis e Mycobacterium leprae) e do fungo Paracoccidioides brasiliensis na bolsa jugal. Comparada com as lesões provocadas em outras localizações e, à exceção do BCG, as lesões induzidas na bolsa são menores e de maior duração e, mesmo quando granulomatosas, incapazes de controlar a multiplicação do agente; nos casos em que houve o desenvolvimento da resposta imune, ele se fez tardiamente e foi acompanhado pela redução do número de parasitas nas lesões. Essas observações apontam a bolsa jugal do hamster como um local de escolha para o estudo sobre a participação da resposta imune no desenvolvimento e modulação das doenças infecciosas e dos granulomas.

Histological evaluation of the lesion induced by inoculation of Leishmania mexicana in the cheek pouch of the hamster

No presente estudo, investigamos o papel da resposta imune na morfologia do granuloma leishmaniótico induzido na bolsa jugal do hamster, um local imunologicamente privilegiado, após inoculação de 3x10(5) Leishmania mexicana. Os animais foram avaliados histológica e imunologicamente até os 120 dias da inoculação. Independente da época do sacrifício, os animais foram sempre não reatores ao teste do coxim plantar. Histologicamente, a inoculação de Leishmania mexicana na bolsa jugal resultou na formação de abcesso que evoluiu para reação granulomatosa rica em formas amastigotas e, posteriormente, para resolução. Esses resultados sugerem que o desenvolvimento da resposta imune não é preponderante no controle da infecção induzida pela Leishmania mexicana inoculada subcutaneamente na bolsa jugal do hamster. Sugerem ainda que os macrófagos que compõe os granulomas leishmanióticos são capazes de eliminar esse parasita, independente da presença de resposta imune avaliável pelo teste do coxim plantar.

Experimental dermatophytosis in hamsters inoculated with Trichophyton mentagrophytes in the cheek pouch

Esse estudo apresenta os resultados obtidos quando da inoculação de Trychophyton mentagrophytes na bolsa jugal do hamster, local imunologicamente privilegiado. Foram utilizados 42 animais: 21 inoculados com 10(6) fungos na bolsa jugal (grupo 1) e, 21 inicialmente inoculados com 10(6) fungos no coxim plantar e, 15 dias após, na bolsa jugal com a mesma quantidade fúngica (grupo 2). Os animais foram sacrificados às 20 h, 3, 7, 14, 30, 60 e 120 dias; foram coletadas amostras da bolsa jugal inoculada, e das patas submetidas ao teste do coxim plantar (TCP). Independente do grupo e do tempo de evolução da infecção, os animais não desenvolveram hipersensibilidade tardia avaliada através do TCP. A pré-inoculação de fungos no coxim plantar não alterou a morfologia das lesões induzidas na bolsa jugal. Assim, nos animais do grupo 1 e grupo 2, a introdução do fungo na bolsa jugal, resultou em lesão focal, constituída por infiltrado inflamatório agudo estéril, com formação de abscesso, que evoluiu para reação macrofágica e, posteriormente, para a resolução mesmo na ausência de resposta imune detectável pelo TCP. Nossos resultados indicam que, apesar do importante papel da resposta imune na regressão espontânea da dermatofitose, outros fatores são, também, parte integral da defesa contra esta infecção fúngica.

The ventral striatum of the Syrian hamster

The Syrian hamster, Mesocricetus auratus, was first used in laboratory experiments some fifty years ago in the Middle East, from animals captured in the wild. 1 Since then the Syrian hamster has been domesticated and used extensively in laboratory studies of motivation, includuing reproduction, feeding, aggression and circadian behaviors. 2 In comparison to the rat, the male Syrian hamster is a solitary animal known for its territorial aggression, photoperiodic mating and hoarding behaviors. Many neural circuits controlling reproductive behaviors are now known. 3 While these motivated behaviors have been demonstrated to be regulated by endocrine status there is increasing evidence that dopamine within the nucleus accumbens conveys the rewarding tone of sexual motivation

Relative sensitivity of the corpus luteum of different days of pregnancy to LH-deprivation in the rat and hamster

Deprivation of endogenous LH by LH antiserum (LH A/S) in 6-day pregnant rats did not affect the luteal or serum progesterone within 24 h. LH A/S treatment on day 7 or 8 of pregnancy, however, caused a 70 and 92% reduction in luteal progesterone, respectively, within 24 h. Serum levels of progesterone showed a similar reduction. In the case of pregnant hamster, unlike the rat, there was a significant decrease in progesterone in the serum, luteal and non-luteal compartments whether the A/S was administered on day 4, 5 or 6. There was more than a 10-fold increase in the luteal cholesterol esters within 24 h whether the A/S was given on day 6, 7 or 8 of pregnancy in the rat. Rat corpora lutea of days 6 and 8 of pregnancy reacted in a like manner to LH-deprivation, showing an increased utilization of [U-14C]glucose to form 14CO2 in vitro. In the rat, LH (25 μg NIH-S19) administration in vivo either on day 6 or day 8 of pregnancy, caused within 2 h an increase in serum and non-luteal progesterone, but luteal progesterone was unchanged. On the other hand, LH administration to hamsters on day 8 of pregnancy caused an increase in progesterone levels in serum, luteal and non-luteal tissue. Incubation of corpora lutea isolated from untreated 6- and 8-day pregnant rats with LH brought about an increase in progesterone secretion into the medium in both cases. The results show that, even though LH-deprivation does not apparently affect progesterone concentration in the corpus luteum of 6-day pregnant rats, it does affect other metabolic parameters such as glucose utilization and cholesterol turnover, suggesting that the corpus luteum of early pregnancy exhibits a continuous dependency on LH for the maintainence of metabolic functions.

Examination of the role of FSH in periovulatory events in the hamster

he need for endogenous FSH in the periovulatory events such as oocyte maturation, ovulation, luteinization, maintenance of luteal function and follicular maturation was examined in the cyclic hamster. A specific antiserum to ovine FSH, shown to be free of antibodies to LH and to cross-react with FSH of the hamster, was used to neutralize endogenous FSH at various times. Administration of this antiserum during pro-oestrus did not affect oocyte maturation and ovulation, as judged by the normality of the ova to undergo fertilization and normal implantation. It also had no effect on the process of luteinization or on the maintenance of luteal function, as indicated by the normal levels of plasma and luteal progesterone during pro-oestrus and oestrus during the cycle and in pregnancy. All these processes were, however, disrupted by administration of an antiserum to ovine LH, thereby demonstrating their dependence on endogenous LH. Although FSH antiserum given at pro-oestrus did not prevent the imminent ovulation, it blocked the ovulation occurring at oestrus of the next cycle. This antiserum was effective in preventing the ensuing ovulation when given at any other time of the cycle until the morning of pro-oestrus. It is concluded that, in the hamster, high levels of FSH during pro-oestrus and oestrus are required for initiating maturation of a new set of follicles which are dependent on the trophic support of FSH throughout the cycle until the morning of pro-oestrus. Such follicles then appear to need only LH for subsequent ovulatory and associated processes.

Succinate and malate improve development of hamster eight-cell embryos in vitro: Confirmation of viability by embryo transfer

The in vitro development of hamster preimplantation embryos is supported by non-glucose energy substrates. To investigate the importance of embryonic metabolism, influence of succinate and malate on the development of hamster 8-cell embryos to blastocysts was examined using a chemically defined protein-free modified hamster embryo culture medium-2 (HECM-2m). There was a dose-dependent influence of succinate on blastocyst development; 0.5 mM succinate was optimal (85.1% ± 3.9 vs. 54.5% ± 3.5). In succinate-supplemented HECM-2m, blastocyst development was reduced by omission of lactate (68.5% ± 7.2), but not pyruvate (85.8% ± 6.2) or glutamine (84.1% ± 2.1). Succinate along with either glutamine or lactate or pyruvate poorly supported blastocyst development (28%-58%). Malate also stimulated blastocyst development; 0.01 mM malate was optimal (86.3% ± 2.8). Supplementation of both succinate and malate to HECM-2m supported maximal (100%) blastocyst development, which was inhibited 4-fold by the addition of glucose/phosphate. The mean cell numbers (MCN) of blastocysts cultured in succinate-supplemented HECM-2m was higher (28.3 ± 1.1) than it was for those cultured in the absence of glutamine or pyruvate (range 20-24). The MCN was the highest (33.4 ± 1.6) for blastocysts cultured in succinate-malate-supplemented HECM-2m followed by those in succinate (28.3 ± 1.1) or malate (24.7 ± 0.5) supplemented HECM-2m. Embryo transfer experiments showed that 29.8% (±4.5) of transferred blastocysts cultured in succinate-malate-supplemented HECM-2m produced live births, similar (P > 0.1) to the control transfers of freshly recovered 8-cells (33.5% ± 2.0) or blastocysts (28.9% ± 3.0). These data show that supplementation of succinate and malate to HECM-2m supports 100% development of hamster 8-cell embryos to high quality viable blastocysts and that non-glucose oxidizable energy substrates are the most preferred components in hamster embryo culture medium. Mol. Reprod. Dev. 47:440-447, 1997. © 1997 Wiley-Liss, Inc.

Micromolar concentration of pentoxifylline improves development in vitro of hamster 8-cell embryos: conrmation of biological viability by embryo transfer

The inßuence of the sperm motility stimulant pentoxifylline (PF) on preimplantation embryo development in hamsters was evaluated. Eight-cell embryos were cultured in hamster embryo culture medium (HECM)-2, with or without PF (0· 0233·6 mM). There was 90%, 37% and 29% inhibition of blastocyst development by 3·6 (used for human sperm), 0·9 and 0 ·45 mM PF, respectively. However, 23 µM PF (exposed to hamster oocytes during IVF) signicantly (P < 0·05) improved blastocyst development (63· 6% v. 51· 8%); morulae development was, however, not curtailed by 0·45 mM or 0·9 mM PF (51·8%±6·0 or 50·5%±11·3, respectively). Post-implantation viability of PF-treated embryos was assessed by embryo transfer; 43% of 80 PF-treated embryos implanted compared with 40% of 79 control embryos. Of the 9 recipients, 6 females delivered pups (19, i.e. 16% of transferred embryos or 53% of implanted embryos). These data show that in hamsters, continuous presence of PF at 0·45-3·6 mM is detrimental to 8-cell embryo development whereas 23 µM PF improves the development of embryos to viable blastocysts which produce live offspring.

Expression of glucose transporter isoforms and the insulin receptor during hamster preimplantation embryo development

During preimplantation development, embryos of many species are known to express up to five isoforms of the facilitative glucose transporter proteins (GLUT). Development of hamster blastocysts is inhibited by glucose. We therefore investigated GLUT isoform and insulin receptor (IR) expression in hamster preimplantation embryos cultured in glucose-free medium from the 8-cell stage onwards. We show that GLUT1, 3 and 8 mRNA are constitutively expressed from the 8-cell to the blastocyst stage. The IR is expressed from the morula stage onwards. Messenger RNA of the insulin-responsive GLUT4 was not detected at any stage. GLUT1 and 3 were localised by immunocytochemistry. GLUT1 was expressed in both embryoblast and trophoblast, in the latter, mainly in basal and lateral membranes directed towards the blastocoel. and embryoblast. GLUT3 was exclusively localised in the apical. membrane of trophoblast cells. We show that hamster preimplantation embryos express several GLUT isoforms thus closely resembling embryos of other mammalian species. Despite endogenous IR expression, the insulin-sensitive isoform GLUT4 was not expressed, indicating that the insulin-mediated glucose uptake known from classical insulin target cells may not be relevant for hamster blastocysts.

Inhibition of Tyrosine Phosphorylation of Sperm Flagellar Proteins, Outer Dense Fiber Protein-2 and Tektin-2, Is Associated With Impaired Motility During Capacitation of Hamster Spermatozoa

In mammals, acquisition of fertilization competence of spermatozoa is dependent on the phenomenon of sperm capacitation. One of the critical molecular events of sperm capacitation is protein tyrosine phosphorylation. In a previous study, we demonstrated that a specific epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor, tyrphostin-A47, inhibited hamster sperm capacitation, accompanied by a reduced sperm protein tyrosine phosphorylation. Interestingly, a high percentage of tyrphostin-A47-treated spermatozoa exhibited circular motility, which was associated with a distinct hypo-tyrosine phosphorylation of flagellar proteins, predominantly of Mr 45,000-60,000. In this study, we provide evidence on the localization of capacitation-associated tyrosine-phosphorylated proteins to the nonmembranous, structural components of the sperm flagellum. Consistent with this, we show their ultrastructural localization in the outer dense fiber, axoneme, and fibrous sheath of spermatozoa. Among hypo-tyrosine phosphorylated major proteins of tyrphostin-A47-treated spermatozoa, we identified the 45 kDa protein as outer dense fiber protein-2 and the 51 kDa protein as tektin-2, components of the sperm outer dense fiber and axoneme, respectively. This study shows functional association of hypo-tyrosine-phosphorylation status of outer dense fiber protein-2 and tektin-2 with impaired flagellar bending of spermatozoa, following inhibition of EGFR-tyrosine kinase, thereby showing the critical importance of flagellar protein tyrosine phosphorylation during capacitation and hyperactivation of hamster spermatozoa.

Examination of the role of follicle stimulating hormone in estrogen biosynthesis Image Image and Image Image in the ovary of cyclic hamster

The effect of neutralizing endogenous follicle stimulating hormone (FSH) or luteinizing hormone (LH) with specific antisera on the Image Image and Image Image synthesis of estrogen in the ovary of cycling hamster was studied. Neutralization of FSH or LH on proestrus resulted in a reduction in the estradiol concentration of the ovary on diestrus-2 and next proestrus, suggesting an impairment in follicular development.Injection of FSH antiserum at 0900 h of diestrus-2 significantly reduced the ovarian estradiol concentration within 6–7 h. Further, these ovaries on incubation with testosterone(T) Image Image at 1600 h of the same day or the next day synthesized significantly lower amounts of estradiol, compared to corresponding control ovaries. Although testosterone itself, in the absence of endogenous FSH, could stimulate estrogen synthesis to some extent, FSH had to be supplemented with T to restore estrogen synthesis to the level seen in control ovaries incubated with T. Lack of FSH thus appeared to affect the aromatization step in the estrogen biosynthetic pathway in the ovary of hamster on diestrus-2. In contrast to this, FSH antiserum given on the morning of proestrus had no effect on the Image Image and Image Image synthesis of estrogen, when examined 6–7 h later. The results suggest that there could be a difference in the need for FSH at different times of the cycle.Neutralization of LH either on diestrus-2 or proestrus resulted in a drastic reduction in estradiol concentration of the ovary. This block was at the level of androgen synthesis, since supplementing testosterone alone Image Image could stimulate estrogen synthesis to a more or less similar extent as in the ovaries of control hamsters.

Studies on follicular growth in the immature rat and hamster: Effect of a single injection of gonadotropin or estrogen on the rate of3H-thymidine incorporation into ovarian DNAin vitro

Initiation of follicular growth by specific hormonal stimuli in ovaries of immature rats and hamsters was studied by determining the rate of incorporation of3H-thymidine into ovarian DNAin vitro. Incorporation was considered as an index of DNA synthesis and cell multiplication. A single injection of pregnant mare serum gonadotropin could thus maximally stimulate by 18 hr3H-thymidine incorporation into DNA of the ovary of immature hamsters. Neutralization of pregnant mare serum gonadotropin by an antiserum to ovine follicle stimulating hormone only during the initial 8–10 hr and not later could inhibit the increase in3H-thymidine incorporationin vitro observed at 18 hr, suggesting that the continued presence of gonadotropin stimulus was not necessary for this response. The other indices of follicular growth monitored such as ovarian weight, serum estradiol and uterine weight showed discernible increase at periods only after the above initial event. A single injection of estrogen (diethyl stilbesterol or estradiol-l7β) could similarly cause 18 hr later, a stimulation in the rate of incorporation of3H-thymidine into DNAin vitro in ovaries of immature rats. The presence of endogenous gonadotropins, however, was obligatory for observing this response to estrogen. Evidence in support of the above was two-fold: (i) administration of antiserum to follicle stimulating hormone or luteinizing hormone along with estrogen completely inhibited the increase in3H-thymidine incorporation into ovarian DNAin vitro; (ii) a radioimmunological measurement revealed following estrogen treatment, the presence of a higher concentration of endogenous follicle stimulating hormone in the ovary. Finally, administration of varying doses of ovine follicle stimulating hormone along with a constant dose of estrogen to immature rats produced a dose-dependent increment in the incorporation of3H-thymidine into ovarian DNAin vitro. These observations suggested the potentiality of this system for developing a sensitive bioassay for follicle stimulating hormone.

Measurement of FSH in the ovarian tissue by radioimmunoassay: Correlation to serum FSH levels and follicular development in the hamster

A method is described for monitoring the concentration of endogenous receptor-bound gonadotropin in the ovarian tissue. This involved development of a radioimmunoassay procedure, the validity of which for measuring all of the tissue-bound hormone has been established. The specificity of the method of measurement was indicated by the fact that high levels of FSH could be measured only in target tissue such as follicles, while non-target organs showed little FSH. Using this method, the amount of FSH in the non-luteal ovarian tissue of the hamster at different stages of the estrous cycle was quantitated and compared with serum FSH levels found at these times. No correlation could be found between serum and tissue FSH levels at all times. On the morning of estrus, for example, when the serum level of FSH was high, the ovarian concentration was low, and on the evening of diestrus-2 the ovary exhibited high concentration of FSH, despite the serum FSH concentration being low at this time. The highest concentration of FSH in the ovary during the cycle was found on the evening of proestrus. Although a large amount of this was found in the Graafian follicles, a considerable amount could still be found in the �growing� follicles. Ovarian FSH concentration could be considered to be a reflection of FSH receptor content, since preventing the development of FSH receptors by blocking initiation of follicular development during the cycle resulted in a decrease in the concentration of FSH in the ovary. The high concentration of FSH in the ovary seen on the evening of diestrus-2 was not influenced either by varying the concentration of estrogen or by neutralization of LH. Neutralization of FSH on diestrus-2, on the other hand, caused a drastic reduction in the ovarian LH concentration on the next day (i.e. at proestrus), thus suggesting the importance of FSH in the induction of LH receptors.

Tyrphostin-A47 inhibitable tyrosine phosphorylation of flagellar proteins is associated with distinct alteration of motility pattern in hamster spermatozoa

To acquire fertilizing potential, mammalian spermatozoa must undergo capacitation and acrosome reaction. Our earlier work showed that pentoxifylline (0.45 mM), a sperm motility stimulant, induced an early onset of hamster sperm capacitation associated with tyrosine phosphorylation of 45-80 kDa proteins, localized to the mid-piece of the sperm tail. To assess the role of protein tyrosine phosphorylation in sperm capacitation, we used tyrphostin-A47 (TP-47), a specific protein tyrosine kinase inhibitor. The dose-dependent (0.1-0.5 mM) inhibition of tyrosine phosphorylation by TP-47 was associated with inhibition of hyperactivated motility and 0.5 mM TP-47-treated spermatozoa exhibited a distinct circular motility pattern. This was accompanied by hypo-tyrosine phosphorylation of 45-60 kDa proteins, localized to the principal piece of the intact-sperm and the outer dense fiber-like structures in detergent treated-sperm. Sperm kinematic analysis (by CASA) of spermatozoa, exhibiting circular motility (at 1st hr), showed lower values of straight line velocity, curvilinear velocity and average path velocity, compared to untreated controls. Other TP-47 analogues, tyrphostin-AG1478 and -AG1296, had no effect either on kinematic parameters or sperm protein tyrosine phosphorylation. These studies indicate that TP-47-induced circular motility of spermatozoa is compound-specific and that the tyrosine phosphorylation status of 45-60 kDa flagellum-localized proteins could be key regulators of sperm flagellar bending pattern, associated with the hyperactivation of hamster spermatozoa.

Effect of follicle-stimulating hormone and its antiserum on the activity of ornithine decarboxylase in the ovary of rat and hamster

The ability of FSH to stimulate the activity of ornithine decarboxylase (ODC) in the ovary of the immature rat and cycling hamster has been examined using specific antisera to gonadotropins. The stimulatory effect of FSH on ODC activity in the ovary of the immature rat was abolished when LH antiserum was administered along with FSH, while similar administration of FSH antiserum had no effect on LH action in stimulating ODC activity, thereby demonstrating the specificity of the LH effect. During the estrus cycle of the hamster, ODC activity in the ovary could be detected only on the evening of proestrus, the maximal activity seen at 1700 h being associated with both the Graafian follicles and the rest of the ovarian tissue. Neutralization of the proestrous FSH surge had no effect on the activity of ODC in either of these tissues, while similar administration of LH antiserum at 1300 h of proestrus completely inhibited the ODC activity in both large follicles and the rest of the ovarian tissue. Thus, the surge of LH, but not of FSH, appears to be responsible for regulating the ODC activity in the ovary of the cycling hamster.