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Basal gonadotropin hormone release rates during the period of selective follicle-stimulating hormone release in the juvenile female rat
Life Sciences, Vol. 45, pp. 863-868 Printed in the U.S.A.
Pergamon Press
JUSAL GoNADmmPIN -RRLRAsRRATRsMlRMGTERPRRIoDoFsFOLLICLR-s-TING mltMa4R RRLRASR IN TEE JuvBHJLEFI@lALEHAT M. Olubunmi Dada, Jordan P. Metcalf* and Charles A. Blake Department of Anatomy, University of South Carolina, School of Medicine, Columbia, South Carolina 29208 and *Department of Anatomy, University of Nebraska Medical Center, Omaha, Nebraska 68105 (Received in final form June 30, 1989)
There are situations in which adult female rats release increased amounts of follicle-stimulating hormone (FSH) independent of increased luteinizing hormone (LH) release. This results from, at least in part, a selective increase in the basal FSH release rate. We investigated whether an increase in the basal FSH release rate is contributory to the rise in serum FSH levels which occurs independent of a rise in serum LH levels in the immature female rat. Rats had high serum FSH concentrations on days 7 and 15 after birth, low serum FSH levels on day 23, and low serum LH levels on all three In contrast, anterior pituitary gland (APG) FSH and LH days. concentrations and contents increased from day 7 to day 15 and the contents increased further from day 15 to day 23. Similarly, basal FSH and LH release rates per mg APG or per APG, as assessed by measurement of FSH and LH released into culture medium containing APG(s) from different aged rats, increased from day 7 to day 15 but did not increase further between days 15 and 23. The results indicate that unlike situations observed to date in adult female rats, a mechanism(s) other than an increase in the basal FSH release rate is involved in selective FSH release in the irmnature female rat. The release of anterior pituitary gland (APG) luteinizing hormone (LH) is stimulated by luteinizing hormone releasing hormone (LHRH) of diencephalic origin (1). Although APG follicle-stimulating hormone (FSH) is also released by LHRH (l-3), there are situations in which serum FSH concentration rises independent of a rise in serum LH concentration. In the female rat, this occurs spontaneously in the immature animal and in the adult cyclic rat during late proestrus and the morning of estrus (3-7). It also occurs experimentally in adult rats ovariectomized on metestrus (8). The spontaneous rise in serum FSH levels in cyclic rats and that which occurs in ovariectomized metestrous rats is due, at least in part, to an increase.in the basal FSH release rate which occurs independent of a change in the basal LH release rate or the APG FSH content (4-8). Changes occur in the APG during the afternoon of proestrus or within hours after ovariectomy on metestrus which result in the gland releasing substantial increased amounts of FSH without further stimulation by diencephalic factors, ovarian secretions, or many other circulating factors as assessed when the glands are placed in organ culture (48). This increase in the basal FSH release rate is likely due to a decreased exposure of the APG to serum inhibin concentrations in vivo because administration of charcoal-extracted porcine follicular fluid which contains inhibin in vivo during proestrous afternoon blocks the increase in the basal FSH 0024-3205189 $3.00 +.OO Copyright (c) 1989 Pergamon Press plc
864
FSH Release in Juvenile Female Rats
vol. 45, No. 10, 1989
release rate (7). These results do not rule out the possibilitiesthat a change in the LHRH release rate, the release of a putative diencephalic factor which selectivelyreleases FSH, and/or some other mechanism could also be involved in causing selectiveFSH release under these situations. However, it is of interest to determinewhether an increase in the basal FSH release rate is involved in all cases where FSH release is increased independentof an increase in LH release. The present study was designed to determinewhether an increase in the basal FSH release rate is involved in the selectiveincrease in serum PSI-I concentration which occurs from the end of the first week to the middle or end of the third week after birth in the immature female rat (9-11). During this period of elevated serum FSH concentrations,serum inhibin levels do not exert a negative feedback effect on FSH secretion (12).
Pregnant, Sprague-Dawley albino rats were purchased from Simonsen Laboratories,Inc. (Gilroy,CA), kept in a temperature-controlled room (24-27'C) with controlledlighting (lights on from 05:OO to 19:00 h daily) and allowed free access to Wayne laboratory rat food and water. Each litter was reduced to eight pups on the day after birth (day 1). Female pups were decapitatedon day 7, 15, or 23. There were 21 rats in each group. Trunk blood was collectedand allowed to clot at room temperaturefor 20 min. The serum was collected and stored at -2O'C until assayed for FSH and LH concentrations. The pituitary gland of each rat was removed and the pars intermediaand pars nervosa were discarded. The entire anteriorpituitary gland (in the case of 7-day-old rats) or right or left hemi-anteriorpituitary glands (in the case of 15- or 23-day-old rats) were weighed and used to measure either the concentrationof FSH and LH in the gland or the basal FSH and LH release rates. Glands used to measure APG FSH and LH concentrations were homogenised immediatelyin 0.02 molar phosphate buffered saline at pH 7.0 and stored frozen at -20°C. Shortly prior to assay, the homogenateswere thawed and centrifu& at 6°C at 1500 X gravity for 15 min to separate supernatantsfrom the pellets of cellular debris. Supernatantswere used to assay for FSH and LH. Glands used to measure basal FSH and LH release rates were placed isxaediately in individual 3 ml vials containing 1.0 ml of medium 199 with unmodifiedRarle's salts containing L-glutamine(Grand Island Biological Co., Grand Island, NY) and buffered to pH 7.4 with sodium bicarbonate. The glands were placed in a Dubnoff metabolic shaker at 37°C with constant exposure to humidified95% 02-5X CO2 for a 30 min preincubation period. The medium was discarded and the vials were washed with fresh medium which was also discarded. Further fresh medium (1.0 ml) was added to each vial and the hemi-or whole glands were incubated for 2 h. Aspirated medium was centrifuged at 1500 X gravity for 10 rainat 6°C and the upper nine-tenths were stored at -20°C until assayed for FSEI and LH concentrations. These concentrationsdivided by the weight of the respective glands or hemi-glands represent the basal FSH or LH release per mg AFG/2 h. FSH and LH were measured by radioimmunoassayusing the materials supplied by the NIADDK. One assay for FSH and one for LH were run for each of the different The intra-assay types of samples; i.e., sera, homogenates end media. coefficientsof variationwere less than 8.2 and 6.1% for the FSH and LB assays, respectively. Single determinationsof serum samples were made. In some cases, there was insufficient sera to measure both gonadotropins in the same serum sample. For both FSH and LH, homogenatesand media were assayed in duplicateand
Vol. 45, No. 10, 1989
FSH Release in Juvenile Female Rats
865
the results were averaged. The volumes of culture medium assayed did not interferewith the quantitativerecovery of the FSH or LH reference preparation (5). Values are expressed in terms of NIADDK rat FSH-RP-1 and LH-RP-1, respectively. The APG FSH or LH concentrationsand the basal FSH or LH release rates per mg APG were multiplied by the weight of the entire APG to obtain the pituitary FSH or LH content and the basal FSH or LH release rates per gland, respectively. Data collected for rats killed at the three different time periods were comparedby analyses of variance. Duncan'smultiple range tests (13) were then performed. A p-value less than 0.05 was consideredstatisticallysignificant. Results Serum FSH concentrations were high (P
866
vol. 45, No. 10, 1989
FSH Release in Juvenile Female Rats
1200 h
1000
0
FSH
m
LH
-c .E
800
ii B
600
P I
400
i? w
200 0 L
AGE
23 15 (days)
FIG. I Mean (2 SRM) serum FSH and LH concentrations in 7-, 15- and 23-day-old female rats. The number of rats in each group was 10 to 17. *P
.z
250
‘.
13
FSH
m
LH
l
5 p
200
”
8 !s
150
”
s “0 I
100..
g
50
:
: ”
0’ 7 AGE
15 (days)
23
FIG. 2 Mean (+ SHM) APG FSH and LH contents in 7-, 15- and 23-day-old female rats. The number of rats in each group was 6 to 7. ** P
Vol. 45, No. 10, 1989
FSH Release in Juvenile Female Rats
15 AGE
867
23
(days)
FIG. 3 Mean (2 SEM) basal FSH and LH release rates per APG in 7-, 15- and 23-dayold female rats. The number of rats was 6 on day 7, 14 on day 15, and 12 on day 23 for both FSH and LH. *** P
LH concentrations and either APG FSH and LH content or basal FSH and LH release rates was evident. Rather, the basal FSH and LH release rates appeared to be related positively to the APG FSH and LH contents, respectively. The trends in APG FSH and LH contents and the basal FSH and LH release rates observed in the present study on days 7, 15 and 23 were similar to the trends observed by Dullart (16) who used rats on days 5, 10, 15, 20 and 25 after birth. Serum FSH concentrations were not measured in that study (16) and thus, it is not possible to assess those data for any contributory role of basal FSH release in the selective increase in serum FSH concentration. It has been suggested that the selective increase in the basal FSH release rate observed in cyclic rats and in ovariectomixed metestrous rats may be due to a decrease in serum inhibin concentration releasing the gonadotrophs from a tonic inhibition which suppresses FSH release (7,17). Such a mechanism would not appear to be operative in 7- and 15-day-old female rats as the administration of anti-inhibin serum to rats within the first two weeks after birth was without effect on serum FSH concentrations (12). It is concluded that a mechanism or mechanisms other than an increase in the basal FSH release rate is involved in causing selective FSH release in the immature female rat. Possibilities include a particular pattern of LHRH release which could preferentially release FSH over LH release. Experimentally, such a mechanism has been shown to be operative in adult female rats (18). Another possibility would be the release of an hypothetical diencephalic factor which releases FSH and not LH (19). In any event, the immature female rat would appear to provide investigators with an excellent model to study selective FSH release controlled by mechanisms other than an increase in the basal FSH release rate.
868
FSH Release in Juvenile Female Rats
Vol. 45, No. 10, 1989
Achlowled~ts This study was supported by a grant from the National Institutes of Health (HD 22687) to CAB. We thank the Pituitary Hormone Distribution Program of the NIADDK for supplying the materials used to assay PSH and LH and Ms. Deborah Barbour and Ms, Carol Able for the typing of the manuscript.
References
1. H. MATSUO, Y. BABA, R.M.G. NAIR, A. ARIMURA and A.V. SCHALLY, Biochem. Biophys. Res. Comm. 42 1334-1339 (1971). 2. C.A. BLAKE, Endocrinology98 461-467 (1976). 3. S.P. KALRA and P.S. KALRA, Endocrine Rev. 4 311-351 (1983). 4. K.A. ELIAS and C.A. BLAKE, Life Sci. 26 749-755 (1980). 5. K.A. ELIAS and C.A. BLAKE, Endocrinology109 708-713 (1981). 6. K.A. ELIAS and C.A. BLAKE, J. Endocrinol.90 345-354 (1981). 7. K.A. ELIAS, R.P. KELCH, H. LIPNER and C.A. BLAKE, Biol. Reprod. -27 1159-1168 (1982). 8. K.A. ELIAS and C.A. BLAKE, Biol. Reprod. 28 1107-1113 (1983). 9. S.R. OJEDA and V.D. RAMIREZ, Endocrinology90 466-472 (1972). 10. K.D. DOHLER and W. WUlTKE, Endocrinology94 1003-1008 (1974). 11. P.C.B. MACKINNON, J.M. MATTOCK and M.B. TER HAAR, J. Endocrinol.70 361-371 (1976). 12. M.D. CULLER and A. NEGRO-VILAR,Molec. Cell. Endocrinol.58 263-273 (1988). 13. D.B. DUNCAN, Biometrics11 l-42 (1955). 14. S.P. KALRA and P.S. KALRA, Endocrinology94 845-851 (1974). 15. C.A. BLAKE, Endocrinology98 445-450 (1973. 16. J. DULLAART, J. Endocrinol. E 309-319 (1977). 17. C.A. BLAKE, In The Anterior Pituitary Gland, A.S. BHATNAGAR, ed.. Raven Press, New York, pp. 227-237 (1983). 18. P.M. WISE, N. RANCE, G.D. BARR and C.A. BARRACLOUGH,Endocrinology-104 940947 (1979). 19. H. MIZUNUMA,WK SAMSON, MD LUMPKIN, SM MCCANN, Life Sci. 22 2003-2009(1983).
Pergamon Press
JUSAL GoNADmmPIN -RRLRAsRRATRsMlRMGTERPRRIoDoFsFOLLICLR-s-TING mltMa4R RRLRASR IN TEE JuvBHJLEFI@lALEHAT M. Olubunmi Dada, Jordan P. Metcalf* and Charles A. Blake Department of Anatomy, University of South Carolina, School of Medicine, Columbia, South Carolina 29208 and *Department of Anatomy, University of Nebraska Medical Center, Omaha, Nebraska 68105 (Received in final form June 30, 1989)
There are situations in which adult female rats release increased amounts of follicle-stimulating hormone (FSH) independent of increased luteinizing hormone (LH) release. This results from, at least in part, a selective increase in the basal FSH release rate. We investigated whether an increase in the basal FSH release rate is contributory to the rise in serum FSH levels which occurs independent of a rise in serum LH levels in the immature female rat. Rats had high serum FSH concentrations on days 7 and 15 after birth, low serum FSH levels on day 23, and low serum LH levels on all three In contrast, anterior pituitary gland (APG) FSH and LH days. concentrations and contents increased from day 7 to day 15 and the contents increased further from day 15 to day 23. Similarly, basal FSH and LH release rates per mg APG or per APG, as assessed by measurement of FSH and LH released into culture medium containing APG(s) from different aged rats, increased from day 7 to day 15 but did not increase further between days 15 and 23. The results indicate that unlike situations observed to date in adult female rats, a mechanism(s) other than an increase in the basal FSH release rate is involved in selective FSH release in the irmnature female rat. The release of anterior pituitary gland (APG) luteinizing hormone (LH) is stimulated by luteinizing hormone releasing hormone (LHRH) of diencephalic origin (1). Although APG follicle-stimulating hormone (FSH) is also released by LHRH (l-3), there are situations in which serum FSH concentration rises independent of a rise in serum LH concentration. In the female rat, this occurs spontaneously in the immature animal and in the adult cyclic rat during late proestrus and the morning of estrus (3-7). It also occurs experimentally in adult rats ovariectomized on metestrus (8). The spontaneous rise in serum FSH levels in cyclic rats and that which occurs in ovariectomized metestrous rats is due, at least in part, to an increase.in the basal FSH release rate which occurs independent of a change in the basal LH release rate or the APG FSH content (4-8). Changes occur in the APG during the afternoon of proestrus or within hours after ovariectomy on metestrus which result in the gland releasing substantial increased amounts of FSH without further stimulation by diencephalic factors, ovarian secretions, or many other circulating factors as assessed when the glands are placed in organ culture (48). This increase in the basal FSH release rate is likely due to a decreased exposure of the APG to serum inhibin concentrations in vivo because administration of charcoal-extracted porcine follicular fluid which contains inhibin in vivo during proestrous afternoon blocks the increase in the basal FSH 0024-3205189 $3.00 +.OO Copyright (c) 1989 Pergamon Press plc
864
FSH Release in Juvenile Female Rats
vol. 45, No. 10, 1989
release rate (7). These results do not rule out the possibilitiesthat a change in the LHRH release rate, the release of a putative diencephalic factor which selectivelyreleases FSH, and/or some other mechanism could also be involved in causing selectiveFSH release under these situations. However, it is of interest to determinewhether an increase in the basal FSH release rate is involved in all cases where FSH release is increased independentof an increase in LH release. The present study was designed to determinewhether an increase in the basal FSH release rate is involved in the selectiveincrease in serum PSI-I concentration which occurs from the end of the first week to the middle or end of the third week after birth in the immature female rat (9-11). During this period of elevated serum FSH concentrations,serum inhibin levels do not exert a negative feedback effect on FSH secretion (12).
Pregnant, Sprague-Dawley albino rats were purchased from Simonsen Laboratories,Inc. (Gilroy,CA), kept in a temperature-controlled room (24-27'C) with controlledlighting (lights on from 05:OO to 19:00 h daily) and allowed free access to Wayne laboratory rat food and water. Each litter was reduced to eight pups on the day after birth (day 1). Female pups were decapitatedon day 7, 15, or 23. There were 21 rats in each group. Trunk blood was collectedand allowed to clot at room temperaturefor 20 min. The serum was collected and stored at -2O'C until assayed for FSH and LH concentrations. The pituitary gland of each rat was removed and the pars intermediaand pars nervosa were discarded. The entire anteriorpituitary gland (in the case of 7-day-old rats) or right or left hemi-anteriorpituitary glands (in the case of 15- or 23-day-old rats) were weighed and used to measure either the concentrationof FSH and LH in the gland or the basal FSH and LH release rates. Glands used to measure APG FSH and LH concentrations were homogenised immediatelyin 0.02 molar phosphate buffered saline at pH 7.0 and stored frozen at -20°C. Shortly prior to assay, the homogenateswere thawed and centrifu& at 6°C at 1500 X gravity for 15 min to separate supernatantsfrom the pellets of cellular debris. Supernatantswere used to assay for FSH and LH. Glands used to measure basal FSH and LH release rates were placed isxaediately in individual 3 ml vials containing 1.0 ml of medium 199 with unmodifiedRarle's salts containing L-glutamine(Grand Island Biological Co., Grand Island, NY) and buffered to pH 7.4 with sodium bicarbonate. The glands were placed in a Dubnoff metabolic shaker at 37°C with constant exposure to humidified95% 02-5X CO2 for a 30 min preincubation period. The medium was discarded and the vials were washed with fresh medium which was also discarded. Further fresh medium (1.0 ml) was added to each vial and the hemi-or whole glands were incubated for 2 h. Aspirated medium was centrifuged at 1500 X gravity for 10 rainat 6°C and the upper nine-tenths were stored at -20°C until assayed for FSEI and LH concentrations. These concentrationsdivided by the weight of the respective glands or hemi-glands represent the basal FSH or LH release per mg AFG/2 h. FSH and LH were measured by radioimmunoassayusing the materials supplied by the NIADDK. One assay for FSH and one for LH were run for each of the different The intra-assay types of samples; i.e., sera, homogenates end media. coefficientsof variationwere less than 8.2 and 6.1% for the FSH and LB assays, respectively. Single determinationsof serum samples were made. In some cases, there was insufficient sera to measure both gonadotropins in the same serum sample. For both FSH and LH, homogenatesand media were assayed in duplicateand
Vol. 45, No. 10, 1989
FSH Release in Juvenile Female Rats
865
the results were averaged. The volumes of culture medium assayed did not interferewith the quantitativerecovery of the FSH or LH reference preparation (5). Values are expressed in terms of NIADDK rat FSH-RP-1 and LH-RP-1, respectively. The APG FSH or LH concentrationsand the basal FSH or LH release rates per mg APG were multiplied by the weight of the entire APG to obtain the pituitary FSH or LH content and the basal FSH or LH release rates per gland, respectively. Data collected for rats killed at the three different time periods were comparedby analyses of variance. Duncan'smultiple range tests (13) were then performed. A p-value less than 0.05 was consideredstatisticallysignificant. Results Serum FSH concentrations were high (P
866
vol. 45, No. 10, 1989
FSH Release in Juvenile Female Rats
1200 h
1000
0
FSH
m
LH
-c .E
800
ii B
600
P I
400
i? w
200 0 L
AGE
23 15 (days)
FIG. I Mean (2 SRM) serum FSH and LH concentrations in 7-, 15- and 23-day-old female rats. The number of rats in each group was 10 to 17. *P
.z
250
‘.
13
FSH
m
LH
l
5 p
200
”
8 !s
150
”
s “0 I
100..
g
50
:
: ”
0’ 7 AGE
15 (days)
23
FIG. 2 Mean (+ SHM) APG FSH and LH contents in 7-, 15- and 23-day-old female rats. The number of rats in each group was 6 to 7. ** P
Vol. 45, No. 10, 1989
FSH Release in Juvenile Female Rats
15 AGE
867
23
(days)
FIG. 3 Mean (2 SEM) basal FSH and LH release rates per APG in 7-, 15- and 23-dayold female rats. The number of rats was 6 on day 7, 14 on day 15, and 12 on day 23 for both FSH and LH. *** P
LH concentrations and either APG FSH and LH content or basal FSH and LH release rates was evident. Rather, the basal FSH and LH release rates appeared to be related positively to the APG FSH and LH contents, respectively. The trends in APG FSH and LH contents and the basal FSH and LH release rates observed in the present study on days 7, 15 and 23 were similar to the trends observed by Dullart (16) who used rats on days 5, 10, 15, 20 and 25 after birth. Serum FSH concentrations were not measured in that study (16) and thus, it is not possible to assess those data for any contributory role of basal FSH release in the selective increase in serum FSH concentration. It has been suggested that the selective increase in the basal FSH release rate observed in cyclic rats and in ovariectomixed metestrous rats may be due to a decrease in serum inhibin concentration releasing the gonadotrophs from a tonic inhibition which suppresses FSH release (7,17). Such a mechanism would not appear to be operative in 7- and 15-day-old female rats as the administration of anti-inhibin serum to rats within the first two weeks after birth was without effect on serum FSH concentrations (12). It is concluded that a mechanism or mechanisms other than an increase in the basal FSH release rate is involved in causing selective FSH release in the immature female rat. Possibilities include a particular pattern of LHRH release which could preferentially release FSH over LH release. Experimentally, such a mechanism has been shown to be operative in adult female rats (18). Another possibility would be the release of an hypothetical diencephalic factor which releases FSH and not LH (19). In any event, the immature female rat would appear to provide investigators with an excellent model to study selective FSH release controlled by mechanisms other than an increase in the basal FSH release rate.
868
FSH Release in Juvenile Female Rats
Vol. 45, No. 10, 1989
Achlowled~ts This study was supported by a grant from the National Institutes of Health (HD 22687) to CAB. We thank the Pituitary Hormone Distribution Program of the NIADDK for supplying the materials used to assay PSH and LH and Ms. Deborah Barbour and Ms, Carol Able for the typing of the manuscript.
References
1. H. MATSUO, Y. BABA, R.M.G. NAIR, A. ARIMURA and A.V. SCHALLY, Biochem. Biophys. Res. Comm. 42 1334-1339 (1971). 2. C.A. BLAKE, Endocrinology98 461-467 (1976). 3. S.P. KALRA and P.S. KALRA, Endocrine Rev. 4 311-351 (1983). 4. K.A. ELIAS and C.A. BLAKE, Life Sci. 26 749-755 (1980). 5. K.A. ELIAS and C.A. BLAKE, Endocrinology109 708-713 (1981). 6. K.A. ELIAS and C.A. BLAKE, J. Endocrinol.90 345-354 (1981). 7. K.A. ELIAS, R.P. KELCH, H. LIPNER and C.A. BLAKE, Biol. Reprod. -27 1159-1168 (1982). 8. K.A. ELIAS and C.A. BLAKE, Biol. Reprod. 28 1107-1113 (1983). 9. S.R. OJEDA and V.D. RAMIREZ, Endocrinology90 466-472 (1972). 10. K.D. DOHLER and W. WUlTKE, Endocrinology94 1003-1008 (1974). 11. P.C.B. MACKINNON, J.M. MATTOCK and M.B. TER HAAR, J. Endocrinol.70 361-371 (1976). 12. M.D. CULLER and A. NEGRO-VILAR,Molec. Cell. Endocrinol.58 263-273 (1988). 13. D.B. DUNCAN, Biometrics11 l-42 (1955). 14. S.P. KALRA and P.S. KALRA, Endocrinology94 845-851 (1974). 15. C.A. BLAKE, Endocrinology98 445-450 (1973. 16. J. DULLAART, J. Endocrinol. E 309-319 (1977). 17. C.A. BLAKE, In The Anterior Pituitary Gland, A.S. BHATNAGAR, ed.. Raven Press, New York, pp. 227-237 (1983). 18. P.M. WISE, N. RANCE, G.D. BARR and C.A. BARRACLOUGH,Endocrinology-104 940947 (1979). 19. H. MIZUNUMA,WK SAMSON, MD LUMPKIN, SM MCCANN, Life Sci. 22 2003-2009(1983).