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Original Studies |
Departments of Obstetrics and Gynecology (W.B., B.R.C., Y.D., W.R.), Urology (W.B.), and Pathology (M.J.B., F.W.) University of Texas Southwestern Medical Center, Dallas, Texas 75235-9032
Address all correspondence and requests for reprints to: William Byrd, Department of Obstetrics and Gynecology, University of Texas Southwestern Medical Center, Dallas, Texas, 75235-9032. E-mail: ebyrd{at}mednet.swmed.edu
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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subunit linked by disulfide
bonds to either a ßA subunit (inhibin A) or a ßB subunit (inhibin
B). The presumed site for the major secretion of inhibin is the Sertoli
cell (6). However, immunocytochemistry studies have shown that the
Leydig cells as well as the Sertoli cells can produce both subunits of
the inhibin B dimer in the pre- and early postnatal testis in humans
and rats (7). Our understanding of the role of inhibins in male physiology has been limited previously by assays that failed to distinguish bioactive dimeric inhibins from unprocessed higher molecular weight inhibin precursors and free subunits of inhibin. The development of specific immunoassays to detect biologically active dimeric inhibin has recently become available (8, 9), allowing a more detailed analysis of the physiological role inhibins play in men and women. Women have measurable levels of both inhibin A and B during the menstrual cycle (10, 11, 12, 13, 14). The circulating levels of inhibin A and B show a diphasic curve, with circulating levels of inhibin B being increased during the follicular phase and inhibin A levels being elevated during the luteal phase. In contrast, men have only measurable amounts of inhibin B in circulation; inhibin A levels are below the detectable limits of the assay (15). Previous studies have demonstrated that men with abnormal semen parameters have less inhibin B when compared with normospermic males. Illingworth et al. (14) first established the negative correlation between FSH and inhibin B in infertile men, normal fertile men, and men with elevated FSH levels.
Although these studies established a role for inhibin B in mature males with spermatogenesis, the role inhibins might play during the development of the testes and initiation of spermatogenesis has not yet been elucidated. Before puberty there is little or no appreciable gonadotropin (FSH and LH) production in the male. There is a brief surge of gonadotropin and testosterone production during the first few months following birth (16, 17). In contrast, the locally produced Mullerian inhibiting substance (MIS), which is made by the Sertoli cells, is secreted in high levels in males from yr 1–4, suggesting that Sertoli cells are active in the absence of any spermatogenesis (18, 19, 20). The levels of inhibins during the prepubertal years have not yet been described.
To evaluate the role of inhibins further, we measured their concentration in males following birth up through adulthood and senescence. Although there was no measurable inhibin A at any time of development, circulating levels of inhibin B increased after birth, remained low from 3–9 yr, and then increased again with the onset of puberty. As part of the aging process there was a gradual decline in circulating inhibin B levels from the mid-40s up to 90 yr of age. In sexually mature males, levels of inhibin B were found to be negatively correlated with the circulating levels of FSH as we previously reported (15). The data presented here demonstrate clear developmental changes in the production of inhibin during infancy, sexual maturation, and senescence in the male. These data also suggest that men with abnormal sperm production or nonobstructive azoospermia have low levels of inhibin B and high levels of FSH when compared with men with normal semen analyses.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Serum samples were separated by centrifugation, aliquoted, and stored at -80 C until analyzed. Seminal plasma was prepared for analysis by centrifugation for 20 min at 2500 x g to pellet spermatozoa, and aliquots of the supernatant were then stored at -80 C. Urine samples were centrifuged at 2500 x g for 20 min to remove particulate matter, aliquoted, and stored at -80 C until analysis.
Study population
Blood samples from adults aged 20–50 yr (n = 171) came from males attending our fertility clinic and fertile men in our artificial insemination program. These blood samples were drawn between 0800 and 1100 h daily. Blood samples from males over 50 yr of age (n = 31) were from men presenting for routine annual physical exam. Blood samples from these males were taken between 0800 and 1600 h. No samples were used if elevated prostrate-specific antigen (PSA) levels (>4 ng/mL) were present. Blood specimens from newborns (cord blood, n = 20), infants, and children (n = 107) were obtained from children who presented at a children’s emergency room. After chart review, samples from children without serious illness underwent a further endocrine screen. All children’s samples used in this study had normal levels of TSH, T3, free T4, T4, testosterone, estradiol, PRL, FSH, LH, and progesterone. Samples were analyzed with the approval of the Institutional Review Board of the University of Texas Southwestern Medical Center.
Inhibin enzyme-linked immunoassays
Aliquots (100 µL) of standards and serum samples were
added to tubes with 50 µL 6% SDS and then incubated at 100 C in a
boiling water bath for 3 min. After cooling, 100 µL assay diluent of
inhibin A or B was added to each tube. This is followed by 50 µL 6%
hydrogen peroxide. The tubes were then incubated for 30 min at room
temperature and centrifuged to separate the gelatinous precipitate. A
96-well microtiter plate (Serotec, Oxford, England) coated with a
monoclonal antibody to the ßA or ßB subunit of inhibin was washed
with inhibin washing buffer and allowed to stand 15 sec before
decanting. Each wash step was repeated four more times before allowing
the wells to drain. An 80-µL aliquot of each treated sample was then
added to the wells. The plate was sealed and then incubated overnight
with constant agitation. The wells were then washed eight times with
inhibin washing buffer and allowed to drain. Following this, 50 µL
alkaline phosphatase conjugated to Fab mouse antihuman inhibin
subunit was added to each well. This plate was then sealed and
incubated for 1–3 h at room temperature with agitation. The wells were
then washed eight times with inhibin washing buffer as above. The plate
was washed an additional three times, each time the inhibin buffer was
allowed to stand for 15 min. After washing, 50 µL substrate solution
containing MgCl2 was added to each well. Following this,
the plate was sealed and incubated at room temperature for 1–2 h with
agitation. After incubation, 50 µL amplifier solution was added, and
then the plate was gently agitated at room temperature and 50
µL stop solution was added to each well to stop the color reaction.
The optical density was measured at 490 nm. The sensitivity of the
assays was 15 pg/mL for inhibin B and 2 pg/mL for inhibin A. Standard
serum samples were run with each assay. Standards were prepared from
patient samples, aliquoted, and stored at -80 C until use. At least
three control standards were run with each inhibin B assay; the
coefficient of variation between assays with these standards ranged
from 3.5 within a plate to 8.6% between plates for inhibin B. There
was less than 1% cross-reactivity between inhibin A and B in this
assay.
Gonadotropin and steroid hormone assays
FSH, LH, and total testosterone were measured on serum samples. All assays were performed using the Immulite immunoassay system (Diagnostic Products Corp., Los Angeles, CA), which is an automated two-site chemiluminescent immunoassay system. The sensitivity of the assasy was was 0.1 mIU/mL for FSH and 0.7 mIU/mL for LH. Inter- and intraassay variations were less than 10%. Serum LH and FSH were determined using the Second International Reference preparation as standards. Serum testosterone was measured with a sensitivity of 0.1 ng/mL.
Semen analysis
Semen analysis was performed, and men were characterized as normal or abnormal based on the World Health Organization Manual, 3rd edition (22). Sperm morphology was based on Kruger’s strict morphology (23).
Statistical analysis
Results are expressed as mean ± SEM. Analysis of group means was performed using ANOVA analysis. Correlation coefficients were investigated for significance by use of Fisher’s P values. P values <0.05 were considered significant.
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Results |
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Serum, seminal plasma free of spermatozoa, and urine were tested
in males aged 20–50 yr with normal sperm production for the presence
of inhibin A and B (Table 1
). Inhibin A
was not detectable (<2 pg/mL) in any of the samples tested. Inhibin B
was not detected in the urine of adult males. Measurable amounts of
inhibin B were found in serum (mean, 196 ± 15 pg/mL) and in the
seminal plasma (1378 ± 340 pg/mL). Levels of inhibin B in the
seminal plasma were high when compared with the serum levels of in the
same male. Seminal plasma samples were collected from six donors after
24, 48, and 72 h of abstinence. The seminal plasma inhibin B
concentrations increased from an average of 527 pg/mL following 24
h of abstinence to 1031 pg/mL at 48 h and 1903 pg/mL at 72 h
of abstinence. In contrast, the seminal plasma from males who had a
previous vasectomy had nondetectable levels of inhibin B
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Serum inhibin B was measured in males from the day of birth up to
90 yr of age (Fig. 1). The data in Fig. 1
excludes adult males who either had been proven to have an abnormal
semen analysis, an abnormal PSA test, or had a serum FSH value greater
than 15 mIU/mL. Following birth there was an increase in inhibin B
during the first year, with the highest peak level (209 ± 41
pg/mL) found in at 3–12 months of age. After this early peak, there
was a decrease in levels of inhibin B after the second year, which
remained low up to the tenth year. Serum levels of inhibin B increased
again with the onset of puberty, beginning in the tenth year. Peak
levels of inhibin B were seen again in the younger adults from 20–29
yr of age. These levels then slowly decreased with increasing age.
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Total serum testosterone and the gonadotropins (FSH and LH) were
measured in the same group of males (Fig. 2). Cord serum had the highest
testosterone levels seen in prepubertal males. Serum testosterone
rapidly declined to barely detectable levels after 1 yr of age and
remained low until puberty. Not until the significant elevation in LH
(Fig. 2B) and FSH (Fig. 2C) observed in males aged 10–14 yr was there
any appreciable increase in the secretion of testosterone. Peak levels
of testosterone were seen in males from 20–29 yr of age. In contrast
to testosterone, there were relatively low levels of the gonadotropins
in cord serum. Following birth there was an elevation of both FSH and
LH that reached a peak from 4–12 months of development. Circulating
levels of gonadotropins then dropped off from 1–10 yr of development.
Although testosterone levels declined in males over 45 yr, there was a
continued increase in FSH and LH, which was paralleled by the decrease
in inhibin B (Fig. 1
).
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This relationship of inhibin B to FSH is shown in Fig. 3, in which inhibin B is plotted with its
corresponding value of FSH. This data represents the serum inhibin B
and FSH values found in all adult males (ages 18–90 yr), regardless of
fertility. There is a negative correlation between the level of FSH and
inhibin B (P = <0.001, r = 0.5). A statistically
significant relationship was not seen with testosterone or LH when
compared with inhibin B. However, if these males are divided into
smaller subsets, there is an interesting pattern of inhibin B
production when compared with FSH, LH, and testosterone. There are two
periods of development in which there was a significant elevation of
serum inhibin B from the baseline, ages 1 day to 1 year and 10–19 yr.
During each of these developmental periods there was a negative
correlation between inhibin B and FSH (P = 0.01 and
0.02, respectively). No statistically significant relationship was
observed between inhibin B levels, LH, and testosterone in these same
age groups. In males ages 2–9 yr, there was no significant
relationship (P > 0.05) between inhibin B
concentrations and levels of FSH, LH, or testosterone. If the males
older than 19 yr were divided into two groups (20–49 yr and 50–90
yr), a negative correlation of inhibin B with FSH is still seen in both
groups (P = <0.001). However, for the first time a
positive correlation was seen between testosterone and inhibin B in
males aged 50 yr and older (P = <0.001).
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Fertile sperm donors, males with normal semen analysis, and men with
isolated defects such as teratozoospermia or asthenozoospermia have
statistically higher levels (P < 0.001) of inhibin B
and lower levels of FSH than males who suffer a defect in sperm
production such as azoospermia or oligospermia. There is no difference
in the testosterone levels in these males. Fertile sperm donors had the
highest sperm production followed by men with normal analysis and men
with an isolated defect of either teratozoospermia or
asthenozoospermia.
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Discussion |
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The maintenance of inhibin B seen in young children aged 3–9 yr after gonadotropin and testosterone levels have gone back to baseline levels is somewhat perplexing. Other workers have shown in GnRH-deficient males that inhibin B secretion is dependent on gonadotropin stimulation of the testis (27, 28). However, studies on gonadotropin-deficient males suggests that once inhibin B secretion is initiated and brought within the normal range, maintenance of inhibin B secretion may occur independently of further gonadotropin stimulation. Adult males with hypogonadotropic hypogonadism may have inhibin B levels at or above levels seen in normal males, again suggesting that once Sertoli cell production of inhibin B is initiated, the maintenance of these levels are independent of gonadotropins (28).
Like inhibin B, MIS is a glycoprotein dimer and a member of the transforming growth factor-ß family. MIS is synthesized by immature Sertoli cells and postnatal granulosa cells (19, 29). Although the activity of MIS on Mullerian structures occurs early in fetal development, the MIS production continues in the male fetus through early childhood. The production of MIS in male children is similar to that seen with inhibin B. MIS in males rises rapidly during the first year of life and then gradually declines until puberty (18, 20). However, MIS does not increase at puberty, as does inhibin B. Although produced by testis until puberty, the physiological importance of MIS has not been determined during postnatal development. A possible diagnostic use for MIS or inhibin measurements would be to measure the presence of active Sertoli cells in children with no palpable testis.
The second cycle of FSH secretion, followed by inhibin B secretion and suppression of FSH production, occurs with the onset of puberty in males. Ideally the onset of puberty and subsequent development of different pubertal stages best described using Tanner staging. Males in this study were not staged, but divided into approximate pubertal stages based on age. Before puberty there are low levels of FSH secretion and a positive, though not statistically significant (P = 0.07) relationship with inhibin B. A recent study on prepubertal and pubertal males has also found that production of inhibin B increases as puberty progresses, and this same shift from a positive to a negative relationship between inhibin B and FSH occurs (30). At this time there is active mitotic division with spermatogonial maturation and accumulation of germ cells undergoing spermatogenesis leading to an increase in testicular size. During this period there is also a dramatic rise in FSH and LH and a sharp increase in the circulating levels of inhibin B. Testosterone secretion lags behind the increase in LH, FSH, and inhibin B. Before the initiation of puberty, the Sertoli cell is the predominant cell type within the seminiferous cords. This contrasts with the appearance in the adult testis in which the germ cells outnumber the Sertoli cells.
In adult males, Inhibin B levels were negatively correlated to FSH levels. Fertile donors and men who produced normal numbers of spermatozoa had significantly higher inhibin B levels and sperm counts and lower FSH values than men with poor sperm production or nonobstructive azoospermia. These data are consistent with the role of inhibin B as an endocrine regulator of FSH secretion and with previous findings in semen donors, fertile, infertile, and hypogonadal men (13, 15, 23, 24, 31).
Reproductive data on men over age 60 yr is limited caused by the number of males that have been evaluated for reproductive function. Although data is scanty, most longitudinal and cross-sectional investigations report a progressive decline in gonadal function; as much as a 30–50% decrease in total and free testosterone in males over age 60 yr (32, 33, 34, 35, 36, 37, 38, 39) and an increase in FSH (40). Other investigators have shown age-related differences in sperm production (37, 41). Johnson et al. (41) demonstrated that there was a gradual decline in the number of Sertoli cells in men and other species during the aging process, which is correlated with an increase in FSH levels and a decrease in sperm production. We have also observed a decline in inhibin B and testosterone levels, and the increase in FSH with males over age 60 yr in this cross-sectional study.
These studies have followed inhibin B throughout the male life cycle. Although the endocrine role of inhibin B in the suppression of FSH secretion has been suggested for years, the possible function that inhibin B plays in the testes in the early stages of life has yet to be determined. Future studies will focus on the endocrine role of inhibin B in the nonproliferative testis.
Received November 11, 1997.
Revised February 19, 1998.
Revised April 14, 1998.
Accepted April 22, 1998.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-ßA dimer) during pregnancy. Clin Endocrinol (Oxf). 42:391–397.[Medline]
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