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S. Villarroya and R. Scholler

Summary. The regional antigenic heterogeneity of human spermatozoa is confirmed with 6 monoclonal antibodies raised against ejaculated human spermatozoa. The topographical localization of the antigenic determinants suggests the existence of at least 6 domains on the human spermatozoon. Different fixatives had severe detrimental effects on the antigen—antibody binding. On live human spermatozoa, each antibody bound to a distinct region: acrosome, equatorial segment, entire tail, neck, midpiece and terminal piece. The antigens detected on the acrosome, equatorial segment and entire tail were surface components, whereas the other three were intracellular structures. The determinant present along the entire tail was a sperm-coating antigen. The molecular weights of the recognized antigens were estimated with the Western blot technique.

Immunostaining of individual ejaculates established that the percentages of positive cells were 12–56% for the acrosome, 8–35% for the equatorial segment, 90–100% for the entire tail, 20–52% for the neck, 9–35% for the midpiece and 36–90% for the terminal piece. In addition, labelling of motile and immotile spermatozoa showed differences in the percentages of positive cells, with 5 out of 6 monoclonal antibodies, or in the fluorescence intensity, with the last one labelling the entire tail.

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S. Villarroya and R. Scholler

Summary. An integral component of human spermatozoa, a glycoprotein of Mr 143 000 (two subunits of Mr 76 000 and 67 000) was recognized by the a-HS 1A.1 monoclonal antibody. The antigen was localized on the plasma membrane over the sperm head, as demonstrated by transmission electron microscopy. The antigen– antibody binding on gametes during changes in their functional state was followed by an indirect immunofluorescence assay of live human spermatozoa. In freshly ejaculated spermatozoa the antibody binding pattern revealed a patchwork quilt-like topography of the plasma membrane over the acrosome; the percentage of positive cells varied from 20 to 78% with a mean of 50% (n = 82). Incubation in a capacitation medium could increase this percentage up to 98%, revealing new epitopes in an energy-dependent and temperature-independent manner; concomitantly, a part of the antigen migrated in energy-independent and temperature-dependent manner and accumulated in a ring over the postacrosome. When an acrosome reaction was induced in vitro in the presence of Ca2+ with either A23187, ionomycin or human follicular fluid, the HS 1A.1 antigen migrated until immobilization in a well defined pattern around the equatorial segment (single band) or around the equatorial and postacrosomal segments (2 or, seldom, 3 bands). The new antigen localization resulted from a lateral diffusion of pre-existing molecules, occurred in only a few minutes, did not require energy and was temperature-dependent. At the same time, the well outlined large patch burst into a multitude of small spots before vanishing. This veil-like labelling was often observed in spermatozoa kept in the seminal plasma or treated with a metabolic poison.

The HS 1A.1 antigen localization reflects surface changes induced by the incubation in a capacitation medium and the acrosome reaction. Apart from the regional heterogeneity of the plasma membrane of a single cell, as noted above, there were differences in the plasma membrane changes in individual spermatozoa from the same ejaculate as well as in semen samples from different donors. The new antibody binding pattern was often alike in successive ejaculates of the same donor. In patients consulting for infertility the percentage of positive cells was often low and migration of the antigen was slight or absent.

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O. M. Møller, M. Mondain-Monval, A. Smith, E. Metzger, and R. Scholler

Summary. During pro-oestrus, baseline LH concentrations for 9 vixens (pooled data) ranged from 0·8 to 5·3 ng/ml. In each vixen, baseline levels were interrupted by elevations of LH ranging from 3·1 to 10·4 ng/ml. A major preovulatory LH surge was detected in all the vixens. The LH peak ranged from 13·5 to 73·0 ng/ml with an average of 27·8 ± 18·8 (s.d.) ng/ml. Plasma LH concentrations declined to a basal level of 1·3 ± 1·0 ng/ml within 48 h of the peak value. The duration of the LH surge was 1–3 days. The LH peak occurred 1 or 2 days before any sexual receptivity was observed. All the vixens were mated twice 2–5 days after the LH peak; 8 conceived. Plasma concentrations of oestradiol-17β increased gradually during the last 6–7 days before oestrus and reached maximum values (124–373 pg/ml) at the time of the preovulatory LH peak. The first significant increase in plasma progesterone concentration occurred simultaneously with the LH peak. During oestrus (normally 3–5 days), progesterone levels rose steeply, attaining a mean concentration of 57·0 ± 17·5 ng/ml when the vixens went out of heat. Androstenedione and testosterone values changed similarly, both increasing at the beginning of pro-oestrus and reaching maximum values (805–1879 pg/ml and 328–501 pg/ml respectively) 1 day before to 1 day after the oestradiol-17β peak.

The electrical resistance of the vaginal tract increased rapidly during the last 2–3 days of pro-oestrus, reaching a maximum value (300–640 Ω) ~2 days after the oestradiol-17β peak that corresponded with the onset of sexual receptivity. Towards the end of oestrus, the values fell to 100–200 Ω.

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M. Mondain-Monval, A. J. Smith, P. Simon, O. M. Møller, R. Scholler, and A. S. McNeilly

Summary. A heterologous radioimmunoassay system developed for the sheep was shown to measure FSH in the plasma of the blue fox. FSH concentrations throughout the year showed a circannual rhythm with the highest values (61 ·6 ± 14·8 ng/ml) occurring shortly before or at the onset of the mating season, a pattern similar to that of LH. The concentration of FSH then declined when androgen concentrations and testicular development were maximal at the time of the mating season (March to May). Thereafter, concentrations remained low (25·2 ± 4·1 ng/ml) in contrast to those of LH. Implantation of melatonin in August and in February maintained high plasma values of FSH after the mating season (142·3 ± 16·5 ng/ml) in association with a maintenance of testicular development and of the winter coat. The spring rise of prolactin was suppressed by melatonin treatment. The release of FSH after LHRH injection was also increased during this post-mating period in melatonin-treated animals, in contrast to the response of the control animals which remained low or undetectable.

These results suggest that changes both in the secretions of FSH and prolactin may be involved in the prolongation of testicular activity and in the suppression of the spring moult after melatonin administration.

Keywords: blue fox; FSH; melatonin; LHRH; seasonal cycle

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A. J. Smith, M. Mondain-Monval, O. M. MØller, R. Scholler, and V. Hansson

Summary. The seasonal changes in testicular weight in the blue fox were associated with considerable variations in plasma concentrations of LH, prolactin, androstenedione and testosterone and in FSH-binding capacity of the testis. An increase in LH secretion and a 5-fold increase in FSH-binding capacity were observed during December and January, as testis weight increased rapidly. LH levels fell during March when testicular weight was maximal. Plasma androgen concentrations reached their peak values in the second half of March (androstenedione: 0·9 ± 0·1 ng/ml; testosterone: 3·6 ± 0·6 ng/ml). A small temporary increase in LH was seen in May and June after the breeding season as testicular weight declined rapidly before levels returned to the basal state (0·5–7 ng/ml) that lasted until December. There were clear seasonal variations in the androgenic response of the testis to LH challenge. Plasma prolactin concentrations (2–3ng/ml) were basal from August until the end of March when levels rose steadily to reach peak values (up to 13 ng/ml) in May and June just before maximum daylength and temperature. The circannual variations in plasma prolactin after castration were indistinguishable from those in intact animals, but LH concentrations were higher than normal for at least 1 year after castration.

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M. Mondain-Monval, O. M. MØller, A. J. Smith, A. S. McNeilly, and R. Scholler

Summary. A heterologous radioimmunoassay system developed for the rabbit and suitable for a wide range of mammalian species has been shown to measure prolactin in the plasma of the blue fox. Evaluation of prolactin levels throughout the year showed that concentrations displayed a circannual rhythm with the highest values occurring in May and June. Prolactin concentrations remained low (∼2·5 ng/ml plasma) from July until April with no consistent changes found around oestrus (March—April). In 8 pregnant females, the prolactin increase in late April and May coincided with the last part of gestation and lactation: concentrations (mean ± s.e.m.) increased to 6·3 ± 0·6 ng/ml at mid-gestation, 9·7 ± 2·1 ng/ml at the end of gestation and 26·7 ± 5·0 ng/ml during lactation. In 10 non-pregnant animals, the mean ± s.e.m. values were 7·2 ± 1·2 ng/ml in April, 8·8 ± 2·2 ng/ml in May and 9·8 ± 1·3 ng/ml in June. The prolactin profile in 4 ovariectomized females was similar to that observed in non-pregnant animals, but the plasma values tended to be lower during the reproductive season (April—June). In intact females, the only large LH peak (average 28 ng/ml) was observed around oestrus. During pro-oestrus, baseline LH levels were interrupted by elevations of 3·1–10·4 ng/ml. During the rest of the year, basal levels were < 3 ng/ml. In ovariectomized females, LH concentrations increased within 2 days of ovariectomy and remained high (35–55 ng/ml) at all times of year.

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A. J. Smith, M. Mondain-Monval, P. Simon, K. Andersen Berg, O. P. F. Clausen, P. O. Hofmo, and R. Scholler

Summary. Bromocriptine administration in the form of slow-release injections to male blue foxes during March–May abolished the normal spring rise in plasma prolactin concentrations seen in May and June. The spring moult was prevented and the treated animals retained a winter coat of varied quality and maturity until the end of the study in August.

Plasma testosterone concentrations fell normally from March until August. Testicular regression was, however, delayed, although there were individual variations in response. Estimation by DNA flow cytometry in early July of the relative numbers of haploid, diploid and tetraploid cells in the testis showed that, in the treated animals, 74–80% of the cells were haploid (maturing germinal cells), 4–6% tetraploid (mainly primary spermatocytes) and the rest diploid cells (somatic cells and the remaining germinal cell types). In the control males, however, no haploid cells were detected and the majority of cells were diploid (93–99%). At castration in August, histological examination revealed various stages of testicular regression in the treated and control animals.

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A. J. Smith, M. Mondain-Monval, K. Andersen Berg, J. O. Gordeladze, O. P. F. Clausen, P. Simon, and R. Scholler

Summary. Testicular weight in young male blue foxes increased steadily from 12 weeks of age (0·4–0·7 g) to reach peak values at the time of the mating season in March–April (5·2–6·6 g), before declining rapidly during May to low values in August at 63 weeks of age (1·3–1 ·6 g). Primary spermatocytes were found in the spermatogenic epithelium at 20 weeks of age and by late December (29 weeks of age) elongated spermatids were seen. There was a good correlation between the seasonal variations in the presence of germ cell types assessed by quantitative analysis of testicular histology and the variations in numbers of haploid, diploid and tetraploid cells measured by DNA flow cytometry: no haploid cells were found before the end of November and peak numbers were observed in March.

Plasma FSH concentrations were increased from December onwards (with the exception of April). There were no clearcut seasonal variations in plasma LH concentrations although values were consistently lower in April. Testosterone concentrations were low for most of the year but increased from the end of January to the middle of April. There was no detectable seasonal variation in LH release in response to LHRH injection, and no typical pattern in plasma FSH concentrations during the first 100 min after injection. Plasma testosterone concentrations after LHRH injection rose gradually during testicular development.

There were large seasonal variations in soluble Mn2+-dependent adenylate cyclase activity in the testis, that paralleled the changes in testicular weight and haploid cell content. Values were low until December and reached a peak at the time of the mating season before falling to basal levels again by June.

The results suggest that immature male blue foxes reach full testicular development (indistinguishable from that of older animals) by the first mating season after birth, at an age of about 40 weeks.

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A. J. Smith, M. Mondain-Monval, K. Andersen Berg, P. Simon, M. Forsberg, O. P. F. Clausen, T. Hansen, O. M. Møller, and R. Scholler

Summary. Melatonin administration to male blue foxes from August for 1 year resulted in profound changes in the testicular and furring cycles. The control animals underwent 5-fold seasonal changes in testicular volume, with maximal values in March and lowest volumes in August. In contrast, melatonin treatment allowed normal redevelopment of the testes and growth of the winter coat during the autumn but prevented testicular regression and the moult to a summer coat the following spring. At castration in August, 88% of the tubular sections in the testes of the controls contained spermatogonia as the only germinal cell type, whereas in the treated animals 56–79% of sections contained spermatids or even spermatozoa. Semen collection from a treated male in early August produced spermatozoa with normal density and motility.

Measurement of plasma prolactin concentrations revealed that the spring rise in plasma prolactin values (from basal levels of 1·6–5·4 ng/ml to peak values of 4·1–18·3 ng/ml) was prevented; values in the treated animals ranged during the year from 1·8 to 6·3 ng/ml. Individual variations in plasma LH concentrations masked any seasonal variations in LH release in response to LHRH stimulation, but the testosterone response to LH release after LHRH stimulation was significantly higher after the mating season in the treated animals, indicating that testicular testosterone production was maintained longer than in the controls.

The treated animals retained a winter coat, of varied quality and maturity, until the end of the study in August.