PROLACTIN RELEASING ACTIVITY IN THE EARLY HUMAN FOETAL HYPOTHALAMUS
A. S. MCNEILLY, D. GILMORE, G. DOBBIE AND T. CHARD * Joint Academic Unit of Obstetrics, Gynaecology and Reproduction Physiology, The Medical Colleges of St Bartholomew's and The London Hospital, 51-53 Bartholomew Close, London, EC1A 7BE and % Department of Physiology, Institute ofPhysiology, The University, Glasgow, G20NA
(Received 17 December 1976) It is generally accepted that the secretion of prolactin from the mammalian pituitary gland is under inhibitory control from the hypothalamus (Meites, 1973). During studies on the development of gonadotrophin secretion in the early human foetus (Gilmore, Dobbie, McNeilly & Mortimer, 1977) it became apparent that up to week 16, the hypothalamus may exert a stimulatory influence over prolactin release which switches to the adult situation as the foetus develops towards term. This communication describes these findings in more detail. Pituitary glands, hypothalami, and cortices were collected from foetuses delivered by hysterotomy between 10 and 19 weeks of pregnancy. The tissues were immediately frozen on solid C02 and stored at -20 °C until extracted. Blood samples were taken from each foetus together with a sample of amniotic fluid and maternal blood. Serum was separated and stored with the fluid at -20 °C until assayed for prolactin as described previously (McNeilly, 1973; McNeilly & Hagen, 1974). Each foetal pituitary gland was extracted into 1ml 0 1 M-HC1, 4 , containing 10"3M-disopropylphosphofluoridate as enzyme inhibitor (Scott & Lowry, 1974). After homogenization for 1 min with a glass rod and sand, the extracts were centri¬ fuged at 2000g for 30 min at 4°C, the supernatant was snap frozen in a mixture of acetonesolid C02 and stored at -20 °C until assayed. Each foetal hypothalamus and cortex was extracted by homogenization in 1ml 0O5M-HC1 (Campbell, Feuer & Harris, 1964), and the extracts were stored at-20°C until assayed. The prolactin releasing or inhibiting activity of the foetal hypothalamic and cortical extracts was assayed by a modification of the technique of McDonald & Gilmore (1971). Mature female Sprague-Dawley rats, maintained under controlled lighting, were ovariectomized 1 month before use. Three days before the test infusions, oestradiol benzoate (50µg) and progesterone (25mg) in corn oil were injected s.c. Extracts (0-9ml) adjusted to pH 7 with NaOH were injected over a period of 10 s into the right carotid artery under light ether anaesthesia. Control infusions of carrier HC1 neutralized ( 7 ) with NaOH as for the extracts, thyrotrophin releasing hormone (TRH) at 200, 100, 20, 10 and 1 ng, dopamine (50ug) or dopamine (50Mg) plus TRH (lOOng) were given under identical conditions. Blood samples (0-2 ml) were with¬ drawn from the iliac vein, before and 15 and 30 min after injection of the test substances. Serum was separated and stored at -20 °C until assayed for prolactin using the rat prolactin radioimmunoassay kit supplied by the NIAMDD. The levels of TRH in 11 hypothalamic and cortical extracts were measured by Dr Jeffcoate of St Thomas's Hospital, London, using a specific radioimmunoassay (Jeffcoate, Fraser, Gunn & White, 1973). Prolactin was detectable in all foetal sera and pituitary glands; the concentra¬ tion in the latter increased with foetal age (Table 1). Levels of prolactin in the amniotic fluid were low and comparable to those in the maternal and foetal circulations at week 10 but increased progressively between weeks 10 and 15 to between 4 and 100 times those in maternal or foetal sera. •[Present address: MRC Unit of Reproductive Biology, 2 Forrest Road, Edinburgh, EHI 2QW.
Table 1. Total content of immunoreactive thyrotrophin releasing hormone (TRH) and bio¬ logical prolactin releasing or inhibitory activity of foetal hypothalamic and cortical extracts during early pregnancy inhibiting activity (ng by radioimmunoassay) (ng TRH equivalents)
Week of
gesta¬ tion
10 10 12 12 13 14 15 15-5 16 17 19
Prolactin levels
Prolactin releasing
TRH content
Foetal
Hypo-
sex
thalamus
F
0-80 1-04 1-24 1-10 1-07
M M M M F M F M F F
1-15 0-60
3-25 1-40 12-80 4-25
or
Cortex 0-63 0-70 0-50
A. S. MCNEILLY, D. GILMORE, G. DOBBIE AND T. CHARD * Joint Academic Unit of Obstetrics, Gynaecology and Reproduction Physiology, The Medical Colleges of St Bartholomew's and The London Hospital, 51-53 Bartholomew Close, London, EC1A 7BE and % Department of Physiology, Institute ofPhysiology, The University, Glasgow, G20NA
(Received 17 December 1976) It is generally accepted that the secretion of prolactin from the mammalian pituitary gland is under inhibitory control from the hypothalamus (Meites, 1973). During studies on the development of gonadotrophin secretion in the early human foetus (Gilmore, Dobbie, McNeilly & Mortimer, 1977) it became apparent that up to week 16, the hypothalamus may exert a stimulatory influence over prolactin release which switches to the adult situation as the foetus develops towards term. This communication describes these findings in more detail. Pituitary glands, hypothalami, and cortices were collected from foetuses delivered by hysterotomy between 10 and 19 weeks of pregnancy. The tissues were immediately frozen on solid C02 and stored at -20 °C until extracted. Blood samples were taken from each foetus together with a sample of amniotic fluid and maternal blood. Serum was separated and stored with the fluid at -20 °C until assayed for prolactin as described previously (McNeilly, 1973; McNeilly & Hagen, 1974). Each foetal pituitary gland was extracted into 1ml 0 1 M-HC1, 4 , containing 10"3M-disopropylphosphofluoridate as enzyme inhibitor (Scott & Lowry, 1974). After homogenization for 1 min with a glass rod and sand, the extracts were centri¬ fuged at 2000g for 30 min at 4°C, the supernatant was snap frozen in a mixture of acetonesolid C02 and stored at -20 °C until assayed. Each foetal hypothalamus and cortex was extracted by homogenization in 1ml 0O5M-HC1 (Campbell, Feuer & Harris, 1964), and the extracts were stored at-20°C until assayed. The prolactin releasing or inhibiting activity of the foetal hypothalamic and cortical extracts was assayed by a modification of the technique of McDonald & Gilmore (1971). Mature female Sprague-Dawley rats, maintained under controlled lighting, were ovariectomized 1 month before use. Three days before the test infusions, oestradiol benzoate (50µg) and progesterone (25mg) in corn oil were injected s.c. Extracts (0-9ml) adjusted to pH 7 with NaOH were injected over a period of 10 s into the right carotid artery under light ether anaesthesia. Control infusions of carrier HC1 neutralized ( 7 ) with NaOH as for the extracts, thyrotrophin releasing hormone (TRH) at 200, 100, 20, 10 and 1 ng, dopamine (50ug) or dopamine (50Mg) plus TRH (lOOng) were given under identical conditions. Blood samples (0-2 ml) were with¬ drawn from the iliac vein, before and 15 and 30 min after injection of the test substances. Serum was separated and stored at -20 °C until assayed for prolactin using the rat prolactin radioimmunoassay kit supplied by the NIAMDD. The levels of TRH in 11 hypothalamic and cortical extracts were measured by Dr Jeffcoate of St Thomas's Hospital, London, using a specific radioimmunoassay (Jeffcoate, Fraser, Gunn & White, 1973). Prolactin was detectable in all foetal sera and pituitary glands; the concentra¬ tion in the latter increased with foetal age (Table 1). Levels of prolactin in the amniotic fluid were low and comparable to those in the maternal and foetal circulations at week 10 but increased progressively between weeks 10 and 15 to between 4 and 100 times those in maternal or foetal sera. •[Present address: MRC Unit of Reproductive Biology, 2 Forrest Road, Edinburgh, EHI 2QW.
Table 1. Total content of immunoreactive thyrotrophin releasing hormone (TRH) and bio¬ logical prolactin releasing or inhibitory activity of foetal hypothalamic and cortical extracts during early pregnancy inhibiting activity (ng by radioimmunoassay) (ng TRH equivalents)
Week of
gesta¬ tion
10 10 12 12 13 14 15 15-5 16 17 19
Prolactin levels
Prolactin releasing
TRH content
Foetal
Hypo-
sex
thalamus
F
0-80 1-04 1-24 1-10 1-07
M M M M F M F M F F
1-15 0-60
3-25 1-40 12-80 4-25
or
Cortex 0-63 0-70 0-50
