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M. J. Harris, M. E. Wallace, and E. P. Evans

Summary. The spontaneous appearance of a Robertsonian translocation in a laboratory colony of genetically wild Peru—Coppock mice gave the opportunity to study potential meiotic nondisjunction soon after the formation of the new chromosome and also in a hitherto untested combination of genotype and environment. Metaphase II scores from the progenitor male had indicated a nondisjunction rate of approximately 10%, a figure that was confirmed by the finding of an estimated 12–16% total trisomic and probable monosomic zygotes in chromosomal studies of Day 9 embryos from heterozygous females. The chromosome studies also showed the presence of a significant excess of normal embryos that were heterozygous for the Robertsonian chromosome.

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R. T. CHATTERTON Jr, J. A. HARRIS, and R. M. WYNN

Summary.

Three specimens were taken from mammary glands of rats killed on the 18th and 21st days of pregnancy and on the 1st day of lactation. Ultrastructural features of the tissue were compared among rats within and between the two stages of development. The similarity among specimens from the same rats made feasible a comparison of serial biopsies obtained every 4 hr, starting on the afternoon of the 21st day of pregnancy. From the 18th to the 21st days of pregnancy, a marked increase in the amount of rough endoplasmic reticulum occurred. The alveolar cells of rats killed on both days and in biopsies obtained at 17 and 13 hr before parturition contained abundant small lipid droplets and vacuoles containing many protein granules with little clear fluid (stasis vacuoles). Alveolar lumina were distended with secretion by 17 hr before parturition. Between 8 and 12 hr before parturition, the accumulated protein and lipid were rapidly extruded from the alveolar cells despite evidence of continued biosynthesis. It is suggested that active transport processes are initiated independently of milk synthesis before parturition.

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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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J. P. HOLLAND, J. M. DORSEY, N. N. HARRIS Jr, and F. L. JOHNSON

Summary.

The effects of l-thyroxine and of surgical thyroidectomy upon the survival of blastocysts have been studied in 200 albino rats. These rats were ovariectomized on the 3rd day of pregnancy, and maintained on progesterone in order to delay implantation. Delay of implantation was confirmed by laparotomy on the 8th day of pregnancy. Subsequent implantation was accomplished by giving 1 μg oestrone daily from the 9th day of pregnancy. At autopsy on the 14th day of pregnancy hyperthyroid rats and hypothyroid rats which had been maintained on daily injections of 2·0 mg progesterone did not differ from their respective control groups in the number of surviving blastocysts. However, hyperthyroid rats which had been maintained on daily injections of 0·4 mg progesterone possessed more implantation sites than controls. Similarly, the hypothyroid rats maintained on daily injections of 0·3 mg progesterone had fewer implantation sites than controls. The experiments suggest that the level of hyperthyroidism tested is beneficial to the maintenance of implantation of delayed blastocysts when low amounts of progesterone are available, while hypothyroidism tends to be detrimental to this process during low progesterone availability.

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N A Czarny, J I Garnham, M S Harris, and J C Rodger

This study describes ovarian changes during the natural and stimulated reproductive cycle of breeding (≤12 month) and retired (>12 month) fat-tailed dunnarts, Sminthopsis crassicaudata. Increased urinary cornified epithelial cells and the influx of leukocytes defined day 0, at which time the naturally cycling females had already ovulated; at day 16 females had no antral follicles, but by day 20 antral follicles had begun to develop. There was no difference between naturally cycling breeding and retired females. Females were stimulated with 1 IU equine serum gonadotropin (eSG) during the intermediate phase on day 16 and killed 3, 4, or 5 days later. Stimulation resulted in a significant increase in the number of growing antral follicles but retired females demonstrated a reduced response. Upon collection from breeding females 4 days following eSG stimulation, 100% of oocytes were at the first polar body (PB1) stage, those collected from retired females were immature upon collection but within 48 h 98.2±1.9% were cultured to the PB1 stage. The rate of ovulation was high in breeding females 5 days following stimulation but retired females were less reliable, and in both groups all oocytes were degraded. This is the first study to describe a reliable technique, involving ovarian stimulation during the intermediate phase and segregation of age groups, allowing the collection of a large number of healthy PB1 stage oocytes from S. crassicaudata. This is important for the development of further assisted reproductive techniques for this species and threatened dasyurids.

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Sarah E Harris, Iris Adriaens, Henry J Leese, Roger G Gosden, and Helen M Picton

Metabolic markers are potentially valuable for assessment of follicle development in vitro. Carbohydrate metabolism of murine preantral follicles grown to maturityover 13 days in vitro has been measured, and metabolism of resulting oocyte–cumulus complexes (OCCs) and denuded oocytes has been compared with in vivo ovulated control counterparts. Spent follicle culture media were analysed for glucose, lactate and pyruvate concentrations. During follicle in vitro growth, glycolysis accounted for a rise from ∼24 to 60% of all glucose consumed. Ovulation induction caused a significant increase in glucose uptake and lactate production by in vitro-grown follicles to 71.7±1.2 and 96.6±4.8 nmoles/day respectively. OCCs grown in vitro had significantly higher rates of glucose consumption and lactate and pyruvate production (110.1± 3.5, 191.8± 8.9 and 31.7± 1.7 pmoles/h respectively) than in vivo ovulated controls (67.4± 8.1, 113.9± 17.1 and 20.2± 4.0 pmoles/h respectively), but a reduced capacity for pyruvate consumption (1.13± 0.06 vs 1.49± 0.06 pmoles/h by in vivo ovulated oocytes). Metabolism of OCCs was affected by the quality of the original follicle. In vitro-grown oocytes had a reduced cytoplasmic volume when compared with controls (168.3± 2.0 vs 199.0± 3.2 proportionately respectively) but a similar rate of metabolism per unit volume. Meiotic status influenced metabolism of both OCCs and denuded oocytes. In conclusion, glucose consumption and lactate production by cultured follicles increased in tandem with developmental progression and were stimulated prior to ovulation. Additionally, the metabolic profiles of in vitro produced OCCs and the oocytes within them are affected by long-term exposure to the culture environment.

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Nicolas M Orsi, Nadia Gopichandran, Henry J Leese, Helen M Picton, and Sarah E Harris

Bovine oocyte maturation in vitro frequently results in abnormal cytoplasmic maturation and failure to acquire developmental competence. This is, in part, likely to be due to the non-physiological nutritional milieu to which oocytes are exposed. Improvements in oocyte developmental potential may be achieved by modelling nutrient profiles on those of preovulatory follicular fluid (FF). However, little is known about fluctuations in FF nutrient levels according to follicle dominance and oestrous cyclicity. This study therefore characterised the carbohydrate and amino acid profile of FF according to these parameters, and compared preovulatory FF composition with that of maturation medium. Carbohydrate concentrations (n = 121) were determined enzymatically whilst amino acid profiles (n = 40) were determined by reverse-phase HPLC. Pyruvate and glucose concentrations were unaffected by follicle dominance, whereas Stage III–IV lactate profiles were higher in non-dominant FF (P < 0.01). While most dominant FF amino acid concentrations were affected by oestrous stage, only glutamate, alanine, leucine and lysine levels fluctuated in non-dominant FF. Glucose and lactate concentrations were significantly negatively correlated, whereas most amino acids were significantly positively correlated with each other. Maturation medium had higher pyruvate and lower lactate concentrations than preovulatory FF (P < 0.001), whereas glucose level was similar. All amino acid levels (except histidine, taurine, alanine and tryptophan) differed significantly between maturation medium and preovulatory FF. These data indicated that FF composition varies throughout the oestrous cycle. Preovulatory FF nutrient profile differed from that of maturation medium, perhaps accounting for the poor developmental competence of in vitro matured oocytes. These data may contribute to the formulation of a nutritionally more physiological maturation medium.