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I. Stoufflet, M. Mondain-Monval, P. Simon and L. Martinet

Summary. Peripheral plasma progesterone concentrations exhibited an increase 10 days before implantation, coinciding with the resumption of blastocyst growth and with a decrease in plasma androgen values (DHA, androstenedione, testosterone). No definite pattern of oestrone was observed and oestradiol concentrations remained undetectable. The production of steroids by dispersed luteal cells showed that the growth of the corpora lutea paralleled that of blastocysts and resulted in hypertrophy followed by hyperplasia of the luteal cell. The production of progesterone in the medium increased with blastocyst size up to implantation; it was enhanced by mink charcoal-treated serum, but prolactin, LH, FSH or a combination of these hormones did not affect the progesterone production, whatever the stage of diapause. DHA and androstenedione secretion increased in the two last stages of blastocyst growth and was enhanced by LH. The conversion of androstenedione and testosterone into oestrone and oestradiol was observed at all stages of embryonic diapause, indicating that corpora lutea contain aromatase activity even at an early stage. The secretion of oestrone was higher than that of oestradiol. The non-luteal tissue contributed up to 50% of the steroid production; while progesterone and androgen production remained constant, that of oestradiol decreased at the end of the delay period. These results indicated a change in the size and the secretory capacity of the luteal cell related to blastocyst development and implantation. Although progesterone was the main product of the corpora lutea, androgens and oestrogens were also secreted.

Keywords: delayed implantation; mink; corpus luteum; steroid hormones; in vivo; in vitro

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I. Wilmut, M. L. Hooper and J. P. Simons

1 AFRC Institute of Animal Physiology and Genetics Research, Roslin, Midlothian EH25 9PS, UK; and2Department of Pathology, University of Edinburgh, Teviot Place, Edinburgh EH8 9AG, UK

Keywords: transgenic animals; gene targeting; reproduction

Contents

Introduction

Methods of genetic manipulation

Microinjection

Retroviral vectors

Embryonal stem cells

Spermatozoa as vectors

Factors affecting the expression of transgenes

Choice of species

Assays for transgene expression

Research strategies using genetically manipulated animals

Ablation of tissues

Cell lineages

Identification of new genes

Modification of gene function

Disease models

Other uses of oncogenes in transgenic animals

Somatic gene therapy

Research in reproduction

Embryogenesis

Homoeobox-containing genes

Genomic imprinting

Hypogonadal mice and the GnRH gene

Development of the reproductive tract

Lactation

Spermatogenesis

Conclusions

Introduction

During the past decade, revolutionary new opportunities in biological research have been created by bringing together techniques developed in two very different areas of biology: molecular biology and mammalian embryo manipulation. A gene from

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M. Caillol, M. Meunier, M. Mondain-Monval and P. Simon

Summary. In the brown hare, fertile mating takes place from the beginning of December to September. Pituitary and ovarian response to a monthly i.v. injection of 5 μg LHRH was studied from September 1983 to October 1984 in 2 groups of 6 hares. The basal concentrations of LH remained undetectable until the end of January, rose from 0·23 ± 0·14 ng/ml from February to a maximum of 1·44 ± 0·57 ng/ml in July. LHRH injection was always followed by a release of LH. Between September and December, the LH value peaked 15 min after injection and returned to basal concentrations 2 h later. From January, this pattern altered and a second peak of LH appeared 2 h after injection. Peak levels 15 min after LHRH were around 10 ng/ml between September and December, increased from 47·0 ± 8·0 ng/ml in January to 106 ± 33 ng/ml in July and decreased in August (69·4 ± 10·6 ng/ml). The values of the second peak rose from 11·0 ± 2·2 ng/ml in January to 90·6 ± 12·4 ng/ml between March and July and decreased in August (24·5 ± 5·1 ng/ml). The LH surge induced by LHRH was always followed by a transient rise in progesterone. During the breeding season, this progesterone secretion increased considerably. Ovulation was possible between January and August and the number of ovulating females was maximum between March and July. The amount and duration of progesterone secretion during the resulting pseudopregnancies increased during the breeding season.

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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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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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S. Harris, M. McClenaghan, J. P. Simons, S. Ali and A. J. Clark

AFRC Institute of Animal Physiology and Genetics Research, Edinburgh Research Station, Roslin, Midlothian EH25 9PS, UK

Keywords: Mammary gland; β-lactoglobulin; transgenic; gene expression

Introduction

The mammary gland is the specialized secretory organ that provides essential nourishment to mammalian young in the form of milk. Milk is primarily composed of water, fats, lactose and proteins; the major protein components are the various caseins and the whey proteins α-lactalbumin, β-lactoglobulin (ruminants) and whey acidic protein (rodents).

Mammary development and milk protein gene expression are regulated by a number of peptide and steroid hormones, as well as cell-cell and cell-substratum interactions within the gland (Topper & Freeman, 1980; Levine & Stockdale, 1985; Li et al., 1987). During gestation the secretory capacity of the mammary gland increases due to cellular proliferation and differentiation; concomitantly milk protein gene expression is initiated in preparation for sustained milk production after parturition. In late lactation milk production

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Claire Glister, Simon J Sunderland, Maurice P Boland, James J Ireland and Phil G Knight

Five isoforms of follistatin (FST) (Mr 31, 33, 35, 37, and 41 kDa) were purified from bovine follicular fluid (bFF). Comparison of their activin and heparan sulphate proteoglycan (HSP) binding properties and biopotencies in the neutralisation of activin A action in vitro revealed that all five isoforms bound activin A, but they did so with different affinities. Only the 31 kDa isoform (FST-288) bound to HSP. FST-288 also showed the greatest biopotency, and the 35 and 41 kDa isoforms were the least potent. To determine whether bovine follicle development is associated with changing intrafollicular FST and activin profiles, we analysed bFF from dominant follicles (DFs) and subordinate follicles (SF) collected at strategic times during a synchronised oestrous cycle. Total FST, activin A and activin AB were measured by immunoassay, whereas individual FST isoforms were quantified by immunoblotting. Follicle diameter was positively correlated with oestrogen:progesterone ratio (r=0.56) in bFF but negatively correlated with activin A (r=−0.34), activin AB (r=−0.80) and ‘total’ FST (r=−0.70) levels. Follicle diameter was positively correlated with the abundance of the 41 kDa isoform (r=0.59) but negatively correlated with the abundance of the 33 and 31 kDa isoforms (r=−0.56 and r=−0.41 respectively). Both follicle statuses (DF and SF) and cycle stage affected total FST, activin A and activin B levels, whereas follicle status, but not cycle stage, affected the abundance of the 41, 37, 33 and 31 kDa FST isoforms. Collectively, these findings indicate that intrafollicular FST isoforms, which differ in their ability to bind and neutralise activins and to associate with cell-surface proteoglycans, show divergent changes during follicle development. Enhanced FST production may play an important negative role, either directly or via the inhibition of the positive effects of activins, on follicle growth and function during follicular waves.

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Michael Garratt, Roslyn Bathgate, Simon P de Graaf and Robert C Brooks

Oxidative stress, overproduction of reactive oxygen species (ROS) in relation to defence mechanisms, is considered to be a major cause of male infertility. For protection against the deleterious effects of ROS, animals have a variety of enzymatic antioxidants that reduce these molecules to less reactive forms. The physiological role of these antioxidants in vivo has been explored extensively through genetic inhibition of gene expression; surprisingly, many of these animals remain fertile in spite of increased oxidative stress. Copper-zinc superoxide dismutase-deficient (Sod1 −/−) male mice are one such example for which in vivo fertility has been repeatedly reported as normal, although examination of fertility has consisted of simply pairing animals of the same strain and checking for litters. This is a fairly low criterion by which to assess fertility. Herein, we show that Sod1-deficient males have zero fertilisation success in sperm competition trials that pit them against wild-type males of an otherwise identical genetic background and are almost completely infertile when mated singly with females of a different genotype. We also show that various aspects of sperm motility and function are impaired in Sod1-deficient mice. Testing the breeding capabilities of mice under more ecologically relevant conditions and with females of different genotypes may help reveal additional physiological causes of infertility.

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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.