Department of Pediatrics, University of California San Francisco, San Francisco, California 94143
THE ONTOGENESIS OF HUMAN FOETAL HORMONES.
II. LUTEINIZING
HORMONE (LH) AND FOLLICLE STIMULATING HORMONE
(FSH)
By Selna L.
Kaplan and Melvin M.
Grumbach
ABSTRACT
The content and concentrations of immunoreactive LH (LER-960) and FSH (LER-869) were determined in 79 human foetal pituitary glands from 68 days of gestation to term, and in the pituitary glands of 3 anencephalic infants, 10 infants and children 2 weeks to 7 years of age, and 4 adults. In addition, the concentration of immunoreactive serum LH, FSH, LHHCG, and HCG was determined in 48 foetuses and 6 anencephalic infants. The mean content of LH in the pituitary glands of the female foetuses increased sharply from 88.2 \m=+-\44.2 ng at 10 to 14 weeks to 4983.8 \m=+-\1128.4 ng at 25 to 29 weeks of gestation. In the male foetuses, the mean content of pituitary LH rose from levels of 21.0 \m=+-\11.6 ng to peak levels of 114.5 \m=+-\57.1 ng at 20 to 24 weeks of gestation. The concentration of LH in the pituitary glands of the children was comparable to that of foetuses at
mid-gestation. The
content of FSH in pituitary glands of female foetuses rose from ng at 10 to 14 weeks to 4788.6 \m=+-\1460.7 ng at 25 to 29 weeks of gestation. In the male foetuses the pituitary content rose from 1.8 \m=+-\0.7 ng at 10 to 14 weeks to 149.5 \m=+-\69.1 ng at 25 to 29 weeks of gestation. The differences in pituitary content of LH and FSH between the male and female foetuses were highly significant from 15 to 29 weeks of gestation. 7.4
mean
\m=+-\5.2
This work was supported in part by grants from the National Institute of Child Health and Human Development and the National Institute of Arthritis, Metabolism, and Digestive Diseases, NIH, USPHS.
The correlation of the incremental changes in the pituitary content of LH and FSH with gestational age was significant (P < 0.02). Immunoreactive serum FSH was detected as early as 84 days of gestation at a concentration of 11.0 ng/ml (LER-869). Serum FSH rose to peak levels (18.2 \m=+-\3.6 ng/ml) at 20 to 25 weeks in a range comparable to that of castrate adults. A decrease was noted by 30 to 34 weeks to levels of 1.7 \m=+-\0.05 ng/ml, comparable to that in cord samples at term (1.8 \m=+-\0.2 ng/ml). A significant negative correlation with gestational age was shown after the mid-gestational period (P < 0.01). Immunoreactive LH, measured by a specific LH assay, was unaffected by levels of HCG present in the foetus. Immunoreactive LH was present in high concentrations by 99 days of gestation (21.5 ng/ml LER-960). Peak levels were attained by 100 to 180 days of gestation with a decrease at them to less than 1.0 ng/ml. A significant negative correlation was noted between serum LH and gestational age. Serum HCG quantified in a \g=b\HCG radioimmunoassay decreased from the relatively high levels in foetuses at 90 to 120 days gestational age to term. The mean concentration in umbilical cord sera was 268.5 \m=+-\161 mIU/ml in contrast to 5400 \m=+-\200 mIU/ml in maternal plasma at term. These data suggest a sex difference in the synthesis and secretion of FSH and LH by the pituitary gland in the human foetus. This sequential pattern of change in the concentration of both serum and pituitary LH and FSH is consistent with the development of a functional hypothalamic pituitary gonadal negative feedback system during foetal life. While the foetal pituitary gonadotrophins do not seem to play a role in the sex differentiation of somatic sex structures in man, the evidence supports an effect on the development of the gonads in females and males and of the differentiated genital tract in the male.
of the ontogenesis of human foetal pituitary gonadotrophins has advanced significantly during the past decade (Levina 1968; Kaplan et al. 1969; Gitlin 8c Biasucci 1969), as well as our understanding of the development of central nervous system regulation of gonadotrophin secretion mediated by luteinizing hormone releasing factor (LRF) (Schally et al. 1973; Blackwell 8c Guillemin 1973; Reichlin 1974), and of the possible role of foetal FSH and LH on the growth and development but not the differentiation of the foetal gonad and somatic sex structure (Grumbach 8c Kaplan 1973; Reyes et al. 1974). The interplay of human chorionic gonadotrophin, a peptide of placental origin and foetal pituitary LH on the maturation of secretory activity of the foetal testis, has not been fully clarified. The synthesis of FSH and LH by the foetal anterior pituitary gland has been demonstrated by histological methods (Pearse 1953; Falin 1961; Mitskevich 8c Levina 1965; Volodina 1966), bioassay (Levina 1968; Rice et al. 1968; Parlow 1974), and immunochemical techniques (Kaplan et al. 1969; Grumbach 8c Kaplan 1973), and secretion by the quantification of serum gonadotrophins (Kaplan el al. 1969; Grumbach 8c Kaplan 1973; Reyes et al. 1974; Levina 1972), and by in vitro studies of the secretion of FSH and LH by
Knowledge
in organ culture (Gitlin 8c Biasucci 1969; Groom et al. 1971; Hartemann et al. 1973; Siler et al. 1972). The radioimmunoassay of LH in foetal serum is complicated by the inability of most anti-LH and anti-HCG sera to discriminate between LH and the placental gonadotrophin HCG. We have attempted to distinguish between LH and HCG by the use of highly specific antisera to the ß subunit of LH or HCG (Vaitukaitis et al. 1972a,b). Quantitation of immunoreactive pituitary and serum gonadotrophins as described in this report and in earlier studies (Levina 1968, 1972; Kaplan et al. 1969; Grumbach 8c Kaplan 1973, 1974; Reyes et al. 1974), including those from this laboratory, indicate sex differences and provide evidence for the maturation of a functional hypothalamic pituitary gonadal feedback system in the human foetus. We are aware of the restrictions in the interpretation of data which are imposed by the measurement of hormone concentration rather than the secretory rate of the pituitary gland and of serum concentration without precise knowledge of possible differences in metabolic clearance rate between foetus and child.
foetal
pituitary glands
MATERIAL AND METHODS
Pituitary glands from 79 foetuses (58 from spontaneous and therapeutic hysterotomy), 3 anencephalic infants, and 10 infants and
abortions and children were obtained at post-mortem examination. Gestational age of the foetuses was estimated from crown-rump measurements and/or from calculations obtained by subtraction of 14 days from the date of onset of the last menstrual period (Shepard 1969). The pituitary gland of the aborted foetuses were quick frozen, stored at —20°C and processed on the day of assay. The pituitary glands from some older foetuses, children, and adults were dissected free of capsular connective tissue, weighed on an analytical balance, and divided into approximately equal sagittal sections to provide tissue for routine pathologic evaluation. The weight of the intact gland and the portion retained for immunologie assay were both obtained within 10 min after removal from the fossa. The pituitary specimens were homogenized in glass tissue grinders in small volumes (0.5 to 1.0 ml) of 0.1 m barbital buffer, pH 8.2 at 4°C on the day the assay was performed. A clear supernatant was obtained following centrifugation of the homogenates at 25 000 r.p.m. for 10 min. The homogenization procedure was altered for the adult pituitary glands by the use of 5 ml of buffer and a more prolonged period of centrifugation (30 min at 4°C). Aliquots of the supernatants were used for immunoassay. Specimens of blood were obtained from 48 of the aborted foetuses, 15 of which had matched pituitary glands; 10 single and 24 matched samples of cord and maternal sera were assayed as well. The quantity of sera available limited the number of different hormone assays carried out on each sample. Immunoassay of LH and FSH was performed by the double antibody methods described previously (Burr et al. 1970; Sizonenko et al. 1970). Iodination of hLH and hFSH was carried out by a modification of the method of Greenwood et al. (1963) followed by separation of the iodinated hormones on Sephadex G75 with 0.15 M phosphosaline buffer at pH 7.8. All values are expressed in terms of purified standards: LER-960 for hLH and LER-869 for hFSH. The conversion factor for LER 907 is 100 ng 1 ng hFSH (LER-869) and 40 ng 1 ng hLH (LER-960). 21 removed at
-
-
The concentration of
serum LH was measured by two methods. In the first, a with 12;'I-/iLH and antisera to /iLH and purified pituitary hLHassay LER-960 as the standard was utilized for the measurement of hLH according to the method of Vaitukaitis et al. (1972«). The cross-reaction of a-LH in this assay system when present in high concentration has not been excluded. Purified HCG cross-reacts to a limited extent, i. e. 50 ng (600 mlU), induces an effect equivalent to 1 ng of purified LH. This amount of HCG is lower than that in diluted foetal or cord sera used in this assay. The higher levels of HCG in maternal sera affect the measurement of LH in this system. The correction factor was derived by determining the degree of displacement of 125I-/iLH from binding to its antiserum induced by increasing concentrations of HCG standard in 4 separate assays. A second method used for the immunoassay of LH was 131I-hLH and antisera to HCG with hLH standard LER-960. This assay does not distinguish between LH and HCG and the values obtained by this assay designated as "LH-HCG" are presented for purposes of comparison. HCG was measured by a homologous /?HCG using l25I-/?HCG and antiserum to /j'HCG (Vaitukaitis cl al. 1972«) supplied by the National Pituitary Agency, and a highly purified Organon HCG standard (11 200 IUAng). In this assay 50 ng of purified LH (LER-960) induces displacement of the 1251-^HCG equivalent to 1 ng of purified HCG. Under these conditions, the concentration of LH in the diluted foetal, cord, and maternal sera tested was not sufficient to affect the results in the assay for HCG. Serum from patients with hypergonadotrophic hypogonadism did not react in the /?HCG assay, but measurable LH was demonstrated in the /iLH assay which supports the specificity of these assays. Serum from patients with hypopituitarism had undetectable levels of FSH, LH, and LH-HCG. Statistical analysis was carried out by logarithmic transformation with a modified Student's ¿-test. Correlation coefficients and analysis of variance and covariance were determined by computer programme according to Snedecor 8c Cochran (1967). The data are presented individually on scattergrams as well as grouped arbitrarily in 4 week intervals for ease of statistical analysis.
homologous /ÎLH
RESULTS
Serial dilutions of foetal pituitary homogenates showed parallelism with the purified standards used for the immunoassay of LH and FSH. In each assay (FSH, /?LH, /5HCG, LH-HCG), serial dilutions of foetal sera showed a parallel degree of displacement of binding of the tracer to that obtained with purified standard. Analysis of the data revealed a significant sex difference in the pituitary content and concentration of LH and FSH. Accordingly, the values are described for male and female foetuses separately at each gestational age period. hFSH in
pituitary glands of female foetuses Immunoreactive FSH was present at 70 days
and
a
mean
concentration of 1.1 content of FSH rose
of age with a content of 3.3 ng in the youngest female foetus available. The ng/mg from 7.4 ± 5.2 (se) ng at 10 to 14 weeks of rapidly
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