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G. F. Weinbauer, J. M. S. Bartlett, U. Fingscheidt, C. G. Tsonis, D. M. de Kretser and E. Nieschlag

Summary. Adult rats (16–18/group) received a single intratesticular injection of 25, 100 or 400 μl glycerol solution (7:3 in distilled water, v/v). Half of the rats in each group were given implants of testosterone, a testosterone-filled Silastic capsule (1·5 cm length) to provide serum values of testosterone within the normal range. After 1 week all animals were killed by decapitation. Serum concentrations of gonadotrophins, testosterone and immunoactive inhibin as well as testicular concentrations of testosterone and bioactive inhibin were determined. Testicular histology was studed in Paraplastembedded tissue stained with PAS and haematoxylin–eosin. Glycerol treatment caused a dose-dependent ablation of spermatogenesis in a distinct area around the site of injection. Serum concentrations of FSH increased proportionally with increasing spermatogenic damage while serum LH and testosterone remained unaltered except with the highest glycerol dose. The rise in serum FSH was significantly correlated with serum (r = −0·70, P < 0·001) and testicular (r = −0·66, P < 0·001) concentrations of inhibin. A less pronounced correlation was found between LH and serum inhibin (r = 0·48). No correlation was found between the concentrations of LH and testicular inhibin or between serum concentrations of FSH and serum testosterone in the 25 and 100 μl groups. Maintenance of low to normal serum testosterone concentrations by means of Silastic implants blocked the elevation of FSH in glycerol-treated animals but failed to affect significantly serum FSH in untreated rats. In all testosterone treated rats testicular inhibin concentrations were markedly reduced in the presence of lowered concentrations (7–14%) of testicular testosterone and unaltered serum FSH concentrations.

These findings indicate that (i) inhibin is involved in the in-vivo regulation of FSH secretion in the adult male rat, (ii) testicular inhibin content, at least in part, may be regulated at the testicular level, and (iii) serum inhibin concentrations to some extent reflect the extent of damage to spermatogenesis and Sertoli cells.

Keywords: inhibin; testosterone; FSH; feedback; testis; rat

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Pedro M Aponte, Takeshi Soda, Katja J Teerds, S Canan Mizrak, Henk J G van de Kant and Dirk G de Rooij

The access to sufficient numbers of spermatogonial stem cells (SSCs) is a prerequisite for the study of their regulation and further biomanipulation. A specialized medium and several growth factors were tested to study the in vitro behavior of bovine type A spermatogonia, a cell population that includes the SSCs and can be specifically stained for the lectin Dolichos biflorus agglutinin. During short-term culture (2 weeks), colonies appeared, the morphology of which varied with the specific growth factor(s) added. Whenever the stem cell medium was used, round structures reminiscent of sectioned seminiferous tubules appeared in the core of the colonies. Remarkably, these round structures always contained type A spermatogonia. When leukemia inhibitory factor (LIF), epidermal growth factor (EGF), or fibroblast growth factor 2 (FGF2) were added, specific effects on the numbers and arrangement of somatic cells were observed. However, the number of type A spermatogonia was significantly higher in cultures to which glial cell line-derived neurotrophic factor (GDNF) was added and highest when GDNF, LIF, EGF, and FGF2 were all present. The latter suggests that a proper stimulation of the somatic cells is necessary for optimal stimulation of the germ cells in culture. Somatic cells present in the colonies included Sertoli cells, peritubular myoid cells, and a few Leydig cells. A transplantation experiment, using nude mice, showed the presence of SSCs among the cultured cells and in addition strongly suggested a more than 10 000-fold increase in the number of SSCs after 30 days of culture. These results demonstrate that bovine SSC self-renew in our specialized bovine culture system and that this system can be used for the propagation of these cells.

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A. Meinhardt, M. K. O'Bryan, J. R. McFarlane, K. L. Loveland, C. Mallidis, L. M. Foulds, D. J. Phillips and D. M. de Kretser

The cellular localization of the activin-binding protein, follistatin, in the rat testis has been a matter of some controversy with different investigators claiming that Sertoli cells, Leydig cells or germ cells are the primary cell types containing this protein. The localization of mRNA encoding follistatin was re-examined using reverse transcription–polymerase chain reaction (RT–PCR) and in situ hybridization as well as the distribution of follistatin by immunohistochemistry. The results demonstrate that mRNA encoding follistatin is located in many germ cells including type B spermatogonia, primary spermatocytes with the exception of the late leptotene and early zygotene stages, and spermatids at steps 1 to 11. It is also found in Sertoli cells and endothelial cells but not in Leydig cells. Immunohistochemistry, using two different antisera to follistatin, showed that this protein was localized to spermatogonia, primary spermatocytes at all stages except the zygotene stage, spermatids at all stages and to endothelial cells and Leydig cells in the intratubular regions. The failure to detect mRNA for follistatin in Leydig cells using RT–PCR and in situ hybridization suggests that the immunohistochemical localization in these cells reflects binding of follistatin produced elsewhere. The widespread localization of follistatin, taken together with its capacity to neutralize the actions of activin, may indicate that follistatin modulates a range of testicular actions of activin, many of which remain unknown.

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A Cesari, M R Katunar, M A Monclus, A Vincenti, J C de Rosas and M W Fornés

Bovine sperm protease, 66 kDa (BSp66) is a serine protease previously characterized in bovine spermatozoa. Like other proteases, it may be present in sperm from other mammalian species different from bovine, playing a role in the fertilization process. In this study, we looked for BSp66 in hamster spermatozoa using heterologous antibodies against bovine BSp66. An immunoreactive protein was detected by Western blotting in mature and immature sperm. The detected protein had two isoforms similar to the ones reported in bovine sperm. Furthermore, indirect immune detection by fluorescence and electron microscopy assays, showed BSp66 signal at the acrosomal region similar to bovine sperm. As it was determined in bovine sperm, the acrosomal reaction displays the antigen within the acrosomal content. When live hamster sperm was incubated with polyclonal antibody against bovine BSp66 a decrease in the number of sperm bound to zona pellucida in homologous IVF and an impairment of head–head agglutination, were observed. These results suggest that a protease homologous to bovine BSp66 is present in golden hamster spermatozoa, with a conserved molecular mass and cellular location. Moreover, hamster BSp66 is probably involved in zona pellucida recognition.

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P. L. A. M. Vos, A. van der Schans, A. A. C. de Wit, M. M. Bevers, A. H. Willemse and S. J. Dieleman

Normally cyclic heifers (n = 34) received 2500 iu pregnant mares' serum gonadotrophin (PMSG) i.m. at day 10 of oestrus, and 15 mg prostaglandin (PG) i.m. at day 12. Thereafter, a monoclonal antibody against PMSG was administered i.v. before (n = 24), at (n = 6) or shortly after (n = 4) the preovulatory LH surge. Peripheral blood concentrations of LH and oestradiol were compared; follicular development was monitored by daily ultrasound scanning; and the numbers of preovulatory-sized follicles and ovulations were counted 96 h after injection of PG following death. Anti-PMSG treatment before the LH surge inhibited the LH surge in 16 heifers (67%). In these heifers, the initial increase in oestradiol concentration upon PMSG stimulation to 167.5 ± 35.0 pmol l−1 was terminated immediately after anti-PMSG treatment and decreased rapidly to basal values, while the number of preovulatory-sized follicles remained constant until 68 h after PG injection; on average 0.4 ± 0.1 ovulations were counted. In the remaining eight heifers, five animals showed an immediate, but temporary, 20–60% drop in oestradiol concentration after anti-PMSG treatment. In all eight heifers 25% of the preovulatory-sized follicles ovulated. Treatment with anti-PMSG at or shortly after the LH surge did not affect the pattern of oestradiol concentration, but a significantly higher ovulation rate was observed in the animals treated shortly after the LH surge: 20.3 ± 2.6 versus 6.3 ± 2.3 in animals treated at the LH surge, which corresponded to 76% and 24% of the preovulatory-sized follicles monitored shortly before the period of multiple ovulations. Thus, neutralization of PMSG at any time before the maximum concentration of the preovulatory LH surge severely suppresses the functionality of the majority or all of the stimulated follicles, which is dependent on the time of injection of anti-PMSG. Although the follicles retain their size, they lose the potential to ovulate. It is concluded that interfollicular asynchrony of development is present at the time the LH surge occurs.

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M D Saenz-de-Juano, F Marco-Jimenez, B Schmaltz-Panneau, E Jimenez-Trigos, M P Viudes-de-Castro, D S Peñaranda, L Jouneau, J Lecardonnel, R Lavara, C Naturil-Alfonso, V Duranthon and J S Vicente

Although numerous studies have demonstrated that cryopreservation alters gene expression, less is known about those embryos that implanted successfully and continued in gestation. To raise the question of the neutrality of this technique, we examine the effects of vitrification through gestation in rabbit before and after the implantation. We monitored the distribution of losses of 569 vitrified morulae, observing that embryos which reach the last pre-implantatory stage are able to implant. However, we found that not all implanted embryos had the ability to continue with their gestation. The results reveal that vitrification decreased foetus and maternal placenta weights at mid-gestation, but led to a higher offspring birth weight. A novel finding is that while no differences in gene expression were detected in pre-implantatory embryos at day 6, vitrification affects a gene and protein expression in the placenta at day 14. Our results for first time reveal strong evidence of modifications in implanted embryos subjected to vitrification, suggesting that the crucial step that vitrified embryos must overcome is the placenta formation. On the basis of these findings, our work leaves the question open as to whether the effects we observed that cause vitrification during foetal development could give rise to some type of physiological or metabolic alteration in adulthood.

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S C Mizrak, F Renault-Mihara, M Párraga, J Bogerd, H J G van de Kant, P P López-Casas, M Paz, J del Mazo and D G de Rooij

Phosphoprotein enriched in astrocytes (PEA-15) is a 15 kDa acidic serine-phosphorylated protein expressed in different cell types, especially in the CN. We initially detected the expression of PEA-15 in primary cultures of Sertoli cells. To assess the presence and localization of PEA-15 in the mouse testis, we studied the expression pattern of the PEA-15 protein by immunohistochemistry and mRNA by in situ hybridization. Both the protein and the mRNA of PEA-15 were localized in the cytoplasm of Sertoli cells, all types of spermatogonia, and spermatocytes up till zygotene phase of the meiotic prophase. Subsequently, with ongoing development of the spermatocytes, the expression decreased and was very low in the cytoplasm of diplotene spermatocytes. To analyze the possible role of PEA-15 in the developing testis, null mutants for PEA-15 were examined. As the PEA-15 C terminus contains residues for ERK binding, we studied possible differences between the localization of the ERK2 protein in wild type (WT) and PEA-15 −/−mice. In the WT testis, ERK2 was localized in the cytoplasm of Sertoli cells, B spermatogonia, preleptotene, leptotene, and zygotene spermatocytes, whereas in the KO testis, ERK2 was primarily localized in the nuclei of these cells and only little staining remained in the cytoplasm. Moreover, in PEA-15-deficient mice, significantly increased numbers of apoptotic spermatocytes were found, indicating an anti-apoptotic role of PEA-15 during the meiotic prophase. The increased numbers of apoptotic spermatocytes were not found at a specific step in the meiotic prophase.

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Y. H. Kim, J. R. McFarlane, G. Almahbobi, P. G. Stanton, P. D. Temple-Smith and D. M. de Kretser

Rat sperm tail fibrous sheath was isolated using mechanical and chemical dissection methods from spermatozoa collected from the cauda epididymis. The procedures used to isolate the fibrous sheath were monitored by phase-contrast microscopy and purity was verified by electron microscopy. SDS-PAGE of isolated total fibrous sheath revealed at least 17 bands when stained with Coomassie brilliant blue and 20 bands with silver stain. The most intensely staining proteins, using both staining methods, were a double band at 80–87 kDa, and a band at 28.5 kDa, whereas with silver staining, bands at 66.2 kDa and 32.7 kDa were also intensely stained. Electroelution following SDS-PAGE was used to isolate 11 of these proteins (116.4, 87.5, 80.9, 66.2, 57.2, 49.7, 46.8, 37.3, 32.7, 28.5 and 15.5 kDa). Amino acid analysis revealed that these proteins were abundant in aspartic and glutamic acid, glycine, serine and leucine, while histidine and phenylalanine were of low abundance. The content of cystine varied widely from 9.4% to 1.4%. The amino termini of the 80.9 kDa, 32.7 kDa, 28.5 kDa and 15.5 kDa proteins were blocked. Immunofluorescence microscopy demonstrated that a polyclonal antiserum to isolated rat fibrous sheath was localized to the principal piece of the rat, rabbit and human spermatozoa, but in the rabbit it also labelled the equatorial region of the head. Western blotting detected all protein bands in isolated fibrous sheath and a similar range of proteins in the spermatozoa of rat and rabbit. Human sperm proteins of 116 kDa and 80 kDa were immunoreactive in common with other species, and there was only weak crossreactivity with the other proteins. These data suggest the presence of common epitopes in the proteins of all three species.

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J. C. Meijer, V. L. Trudeau, F. H. de Jong, M. Bevers, E. N. Noordhuizen-Stassen and C. J. G. Wensing

Summary. Effects of GnRH, administered via the testicular artery, on testicular steroidogenesis were studied in rams during the non-breeding season. Concentrations of testosterone and 17-hydroxyprogesterone in testicular venous blood showed similar profiles which were identical for GnRH-treated (0·5 ng infused over 60 min or 25 ng injected) and control testes. Increases of testicular venous concentration of both hormones were only marginally reflected in peripheral venous concentrations. Peripheral administration of hCG (200 i.u., i.v.) stimulated testosterone secretion to a larger extent than 17-hydroxyprogesterone secretion in 10/11 rams, GnRH-treated and control testes showing identical responses. High testicular venous concentrations of both hormones after administration of GnRH were paralleled by increased concentrations of endogenous LH. These LH peaks were evoked by 25 ng GnRH in 7/8 rams. The observed effects of GnRH treatment on testicular steroid secretion thus cannot be considered to be the result of direct stimulation of steroidogenesis by GnRH.

Keywords: GnRH; in vivo; testis; ram; testosterone; progesterone

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J. L. Brown, M. Bush, D. E. Wildt, J. R. Raath, V. de Vos and J. G. Howard

We tested the ability of several GnRH analogues to suppress pituitary–testicular activity and potentially musth in free-ranging African elephants (Loxodonta africana). In Study 1, adult bulls were given 4 or 12 mg GnRH antagonist (Detirelix) or saline i.m. on day 0 (n = 3 bulls per treatment). Animals were then recaptured on day 2 (about 48 h later) and given 300 μg GnRH i.v. to assess the ability of the antagonist to block pituitary activity. Detirelix reduced (P < 0.05) basal concentrations of serum LH and testosterone on day 2 compared with day 0, with no effect of dose. Similarly, LH and testosterone release induced by GnRH were also reduced (P < 0.05) in the Detirelix-treated bulls (50–70% reduction in peak concentrations). In Study 2, elephants were given 30 mg of a structurally similar GnRH antagonist (103-201-40; n = 6), 22.5 mg of a long-acting GnRH agonist (Lupron Depot; n = 4) or D-mannitol carrier (n = 4) i.m. on day 0. All bulls were recaptured and given GnRH on day 2 (103-201-40 treatment) or on days 2 and 20 (Lupron Depot treatment) after the initial injection. In contrast to Detirelix, 103-201-40 did not inhibit basal or GnRH-induced LH or testosterone secretion. Pituitary–testicular responses to Lupron Depot were initially stimulatory, as evidenced by increased (P < 0.05) LH and testosterone secretion on days 0 and 2. By day 20, basal LH concentrations had returned to baseline values and the response to GnRH was markedly reduced (P < 0.05), indicating that the pituitary was at least partially desensitized. Basal testosterone concentrations had also returned to baseline values by day 20 after Lupron Depot treatment. However, despite the attenuated LH response to GnRH, subsequent testosterone secretion was increased (P < 0.05) compared with controls, suggesting the testes of agonist-treated bulls had, instead, become hyper-responsive to small increases in LH secretion. These results suggest that GnRH analogues can suppress the pituitary–gonadal axis in African elephants; however, longer treatment periods, more frequent injection intervals or higher doses are probably needed to inhibit testosterone secretion completely and, thus, musth.