Hormone Research Laboratory, University of California, San Francisco, California 94143, USA
THE USE OF ANTISERUM TO RAT PROLACTIN TO EVALUATE ITS ROLE AS LUTEOTROPHIN IN THE HAMSTER
By M. R. Sairam, A.
Jagannadha
Rao and Choh Hao Li
ABSTRACT Rabbit antisera to rat
prolactin was found to cross-react with the hamster pituitary extract by double diffusion and quantitative precipitin techniques. Short term neutralization of prolactin by antiserum to rat prolactin either in early or late stages of gestation did not have any deleterious effects. Prolonged administration of rat prolactin antiserum resulted in an increase in the incidence of resorptions, but pregnancy was still maintained followed by normal parturition. This is in marked contrast to the abortive effects of an antiserum of ICSH. It was concluded that prolactin does not play a major role in maintaining pregnancy of the hamster. been known that prolactin is a luteotrophin in the rat (Evans et al. 1941; Astwood 1941). This was primarily derived from the observations that prolactin is capable of maintaining dioestrus and pseudopregnancy in the hypo¬ physectomized animal (Evans et al. 1941; Astwood 1941; Macdonald 8c Greep 1968; Greenwald Sc Rothchild 1964). Recently, reports have shown that while ICSH1) plays an important role in luteal maintenance, its activity is synergistic with prolactin (Morishige Sc Rothchild 1974; Raj et al. 1974). It appears to us that the ability to maintain pregnancy is a better criterion for luteotrophic It has
long
·) Abbreviations: ICSH, interstitial cell stimulating hormone; PRL, , hamster pituitary equivalent. serum;
prolactin; A/S,
anti¬
examine prolactin's role in main¬ taining pregnancy by selective elimination of circulating hormone in the preg¬ nant hamster using rabbit antisera to rat prolactin.
functions. In the present investigation,
we
MATERIALS AND METHODS
Endocrinology Study Section, National Institutes of was isolated according to the method pre¬ viously described (Papkoff et al. 1965). Rabbit antisera to rat prolactin and ovine ICSH were prepared according to published procedures (Moudgal 8- Li 1961). The double diffusion technique of Ouchterlony (1953) and the quantitative precipitin pro¬ cedure of Heildeberger and Kendall as outlined by Rabat Se Mayer (1948) was em¬ ployed to characterize the antiserum. Hamsters were purchased from the local dealer and mated with confirmed fertile males. The day of insemination was considered day 1 of pregnancy; pregnancy was confirmed by exploratory laparotomy on day 6. Animals treated with normal rabbit serum (NRS) served as controls. All injections were given subcutaneously and the animals were examined daily for external vaginal bleeding. Autopsies were performed on the 14th or 15th day of pregnancy and uterine horns were examined for the presence of live and resorbed foetuses. The ovaries and mammary glands were dis¬ sected free of fat, dried on filter paper, and weighed in a torsion balance. Rat
prolactin
was a
gift
from the
Health, Bethesda, Maryland. Ovine ICSH
Agar gel serum
to
Fig.
L
diffusion pattern of rat prolactin and hamster pituitary extract with anti¬ rat prolactin (center well 75 «1): wells 1 and 3, rat prolactin (25 /(g); wells 2 . and 4, hamster pituitary extract 1/10
RESULTS
As shown in Fig. 1, the antiserum to rat prolactin gave a single precipitin line with homologous antigen, but it showed a partial identical precipitin band with the hamster pituitary extract by the double diffusion technique (Ouchterlony 1953). When the quantitative precipitin procedure (Kabat Sc Mayer 1948) was utilized, the antiserum cross-reacted with the hamster pituitary extract to the extent of 47 */o in comparison with the quantitative precipitin curve of rat prolactin (Fig. 2). From these data it was estimated that one ml of the anti¬ serum could neutralize approximately 110 fig of hamster prolactin. Table 1 shows that when one ml of antiserum was administered daily be¬ ginning on day 6 and continued through day 8, no deleterious effects on the course of the pregnancy were noticed; also, there was no reduction in the ovarian or mammary gland weight. Even when daily injections (1 ml) of the antiserum were extended to day 14, no drastic effects on the course of preg¬ nancy occurred (Group IV, Table 1); however, three of the five animals showed slight external vaginal bleeding on day 10 of pregnancy, which later subsided. On autopsy, all the animals had a few resorbed sites, but out of a total of 45 sites, only 12 were resorbed with mean ovarian weight comparable to that of the NRS treated group (Group III, Table 1). A significant reduction in the mammary gland weight was also noticed (Group IV, Table 1). In con-
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2
120
THE USE OF ANTISERUM TO RAT PROLACTIN TO EVALUATE ITS ROLE AS LUTEOTROPHIN IN THE HAMSTER
By M. R. Sairam, A.
Jagannadha
Rao and Choh Hao Li
ABSTRACT Rabbit antisera to rat
prolactin was found to cross-react with the hamster pituitary extract by double diffusion and quantitative precipitin techniques. Short term neutralization of prolactin by antiserum to rat prolactin either in early or late stages of gestation did not have any deleterious effects. Prolonged administration of rat prolactin antiserum resulted in an increase in the incidence of resorptions, but pregnancy was still maintained followed by normal parturition. This is in marked contrast to the abortive effects of an antiserum of ICSH. It was concluded that prolactin does not play a major role in maintaining pregnancy of the hamster. been known that prolactin is a luteotrophin in the rat (Evans et al. 1941; Astwood 1941). This was primarily derived from the observations that prolactin is capable of maintaining dioestrus and pseudopregnancy in the hypo¬ physectomized animal (Evans et al. 1941; Astwood 1941; Macdonald 8c Greep 1968; Greenwald Sc Rothchild 1964). Recently, reports have shown that while ICSH1) plays an important role in luteal maintenance, its activity is synergistic with prolactin (Morishige Sc Rothchild 1974; Raj et al. 1974). It appears to us that the ability to maintain pregnancy is a better criterion for luteotrophic It has
long
·) Abbreviations: ICSH, interstitial cell stimulating hormone; PRL, , hamster pituitary equivalent. serum;
prolactin; A/S,
anti¬
examine prolactin's role in main¬ taining pregnancy by selective elimination of circulating hormone in the preg¬ nant hamster using rabbit antisera to rat prolactin.
functions. In the present investigation,
we
MATERIALS AND METHODS
Endocrinology Study Section, National Institutes of was isolated according to the method pre¬ viously described (Papkoff et al. 1965). Rabbit antisera to rat prolactin and ovine ICSH were prepared according to published procedures (Moudgal 8- Li 1961). The double diffusion technique of Ouchterlony (1953) and the quantitative precipitin pro¬ cedure of Heildeberger and Kendall as outlined by Rabat Se Mayer (1948) was em¬ ployed to characterize the antiserum. Hamsters were purchased from the local dealer and mated with confirmed fertile males. The day of insemination was considered day 1 of pregnancy; pregnancy was confirmed by exploratory laparotomy on day 6. Animals treated with normal rabbit serum (NRS) served as controls. All injections were given subcutaneously and the animals were examined daily for external vaginal bleeding. Autopsies were performed on the 14th or 15th day of pregnancy and uterine horns were examined for the presence of live and resorbed foetuses. The ovaries and mammary glands were dis¬ sected free of fat, dried on filter paper, and weighed in a torsion balance. Rat
prolactin
was a
gift
from the
Health, Bethesda, Maryland. Ovine ICSH
Agar gel serum
to
Fig.
L
diffusion pattern of rat prolactin and hamster pituitary extract with anti¬ rat prolactin (center well 75 «1): wells 1 and 3, rat prolactin (25 /(g); wells 2 . and 4, hamster pituitary extract 1/10
RESULTS
As shown in Fig. 1, the antiserum to rat prolactin gave a single precipitin line with homologous antigen, but it showed a partial identical precipitin band with the hamster pituitary extract by the double diffusion technique (Ouchterlony 1953). When the quantitative precipitin procedure (Kabat Sc Mayer 1948) was utilized, the antiserum cross-reacted with the hamster pituitary extract to the extent of 47 */o in comparison with the quantitative precipitin curve of rat prolactin (Fig. 2). From these data it was estimated that one ml of the anti¬ serum could neutralize approximately 110 fig of hamster prolactin. Table 1 shows that when one ml of antiserum was administered daily be¬ ginning on day 6 and continued through day 8, no deleterious effects on the course of the pregnancy were noticed; also, there was no reduction in the ovarian or mammary gland weight. Even when daily injections (1 ml) of the antiserum were extended to day 14, no drastic effects on the course of preg¬ nancy occurred (Group IV, Table 1); however, three of the five animals showed slight external vaginal bleeding on day 10 of pregnancy, which later subsided. On autopsy, all the animals had a few resorbed sites, but out of a total of 45 sites, only 12 were resorbed with mean ovarian weight comparable to that of the NRS treated group (Group III, Table 1). A significant reduction in the mammary gland weight was also noticed (Group IV, Table 1). In con-
160r140
2
120
