# Prolactin and Male Fertility: The Long and  Short  Feedback Regulation

Authors: M. K. Gill-Sharma
Journal: International Journal of Endocrinology
 (2009)

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## Abstract

In the last 20 years, a pituitary-hypothalamus tissue culture system with intact neural and portal connections has been developed in our lab and used to understand the feedback mechanisms that regulate the secretions of adenohypophyseal hormones and fertility of male rats. In the last decade, several in vivo rat models have also been developed in our lab with a view to substantiate the in vitro findings, in order to delineate the role of pituitary hormones in the regulation of fertility of male rats. These studies have relied on both surgical and pharmacological interventions to modulate the secretions of gonadotropins and testosterone. The interrelationship between the circadian release of reproductive hormones has also been ascertained in normal men. Our studies suggest that testosterone regulates the secretion of prolactin through a long feedback mechanism, which appears to have been conserved from rats to humans. These studies have filled in a major lacuna pertaining to the role of prolactin in male reproductive physiology by demonstrating the interdependence between testosterone and prolactin. Systemic levels of prolactin play a deterministic role in the mechanism of chromatin condensation during spermiogenesis.


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## Body

## 1. Introduction

Male fertility in mammals is regulated by the two
adenohypophyseal hormones, luteinizing hormone (LH) and follicle stimulating
hormone (FSH), through synthesis of testosterone in the interstitial cells of
Leydig and its aromatization to estradiol in the Sertoli cells of the testis.
The regulated release of the hypothalamic gonadotropin-releasing hormone (GnRH)
ensures normal functioning of the hypothalamo-hypophysio-gonadal axis, through
secretion of gonadotropins and testosterone in systemic circulation, necessary
for spermatogenesis, maturation of spermatozoa, and reproductive behaviour
[1, 2]. Prolactin, a 23 Kd hormone, synthesized in the adenohypophyseal
lactotrophs, has no known target organ or defined role in male reproduction. Yet,
expression of prolactin receptors on choroid plexuses and hypothalamus
presupposes a latent role for this hormone in the regulation of male fertility
[3, 4]. In particular, advancement of knowledge in the area of prolactin
signaling cascades across species suggests a conserved physiological role from
rodents to humans [5]. Although the functional significance of prolactin to
male reproduction has not been unequivocally established, the hormone has been
associated primarily with male infertility. Acute hyperprolactinemia is known
to suppress testosterone synthesis and male fertility through prolactin-induced
hypersecretion of adrenal corticoids or by inhibiting the secretion of GnRH
through prolactin receptors on hypothalamic dopaminergic neurons [6, 7]. Dopamine
has been implicated in the release of endorphins from opiatergic neurons that
inhibit the secretion of GnRH [8].Since a clear consensus about the involvement of
prolactin in male reproduction has not emerged even from genetically engineered
mouse models wherein prolactin signaling has been disrupted, not enough
attention has been focused on the regulation of its secretion [9, 10]. However,
the absence of detrimental effects on male fertility in prolactin receptor-deficient
mice does not preclude a role for prolactin, but rather indicates the existence
of compensatory mechanisms reported in literature for certain genetically
modified mouse models [11, 12]. Albeit, genetically modified mice overexpressing
prolactin, could prove to be alternative models in understanding the
physiological role of this hormone in modulating male fertility [13]. A host of
releasing and release-inhibiting factors of pituitary and hypothalamic origin
have been suggested to be involved in prolactin secretion but their physiological
relevance in male reproduction remains a moot point. Dopamine remains the only
acceptable negative regulator of prolactin secretion of physiological relevance
[14]. Prolactin primarily regulates its own release via a short feedback loop
through autoreceptors on the hypothalamic tuberoinfundibular dopaminergic
neurons (TIDA), which release dopamine (DA) into the long portal vessels [15].
Dopamine reaches the adenohypophysis through pituitary portal circulation where
its inhibitory effects are mediated via cognate D2 dopaminergic receptors on
the lactotrophs [16]. Among the gonadal steroids, high estradiol levels appear
to render the dopaminergic neurons refractory to prolactin autofeedback making
it improbable to reverse the effects of physiological or pathological
hyperprolactinemia [17–19]. In such an eventuality, it is tempting to assume
that additional androgen-dependent feedback mechanisms might be involved in
restraining the secretion of prolactin, which has a propensity for
hypersecretion [20].Conventionally, prolactin is without a target organ
and, therefore, not subject to long feedback regulation. However, prolactin
has also been reported to play a role in the synthesis of testosterone through
upregulation of LH receptors on Leydig cells but the relevance of this
mechanism to reproductive physiology has not been understood [21, 22].
Testosterone is known to be involved in the feedback inhibition of LH, the
hormone responsible for its synthesis in the testis [23]. By analogy, it is
again tempting to assume that a similar mechanism of feedback inhibition by
testosterone could also exist for prolactin. Such a mechanism could conceivably
be playing a vital role in male reproductive physiology by modulating prolactin
and testosterone levels in systemic circulation under conditions of mild-to-moderate
hyperprolactinemia arising out of emotional or chemical stress. Thus, in view
of the doubtful significance of prolactin in the regulation of male fertility,
lacunae continue to persist pertaining to mechanisms underlying its secretion.
Towards this end, in vitro and in vivo models developed in our lab have focused
on this aspect. Several novel findings about feedback mechanisms have emerged
from these studies that suggest a modulatory role for prolactin in the
regulation of male fertility.


## 2. Hypothalamo Hypophyseal-Gonadal Factors Underlying Prolactin Secretion

Prolactin secretion is regulated by factors
originating from the hypothalamus, the hypophysis, and the testes in the male
rats. These factors are either inhibitory or stimulatory in action such that
their cumulative effect determines the systemic levels of this hormone. The
hypothalamic input to the pituitary could be in the form of
hypothalamic-inhibitory activity (HPIA) or hypothalamic-releasing activity
(HPRA). The physiological relevance of inhibitory and stimulatory factors
remains to be unequivocally demonstrated. Towards this end, we have used an in
vitro organ culture system comprising the rat hypothalamus and pituitary in
anatomic juxtaposition to establish the existence of complex interactions
between putative factors from both organs. The
development of the rat pituitary-hypothalamus tissue culture (PHc) with intact
neural connections and portal plexus in our lab in 1985 was a major advancement
in reproductive Neuroendocrinology that spawned several studies designed to
understand the feedback regulation of pituitary hormones in vitro [24]. The
hypothalamic island, demarcated by cutting along the lateral hypothalamic
sulci, posterior edge of the optic chiasma, and the anterior edge of the
mammillary bodies, was lifted from its seat on the Sella turcica with an
undercut 2-3 mm in depth,
along with the pituitary attached to it via hypophyseal stalk (see Figure 1).
PHc comprises all the major dopaminergic perikarya viz. Tuberoinfundibular (TIDA),
periventricular (PHDA), tuberohypophyseal (THDA), as well as the long and short
portal vessels [25, 26]. PHc and intact pituitaries (PI) were incubated in
Dulbecco’s Modified Eagle’s Medium (DMEM), fortified with nonessential amino
acids, 3 mM NaHCO3 and 0.1 M HEPES
(4-(2-hydroxyethyl)-piperazine-1-ethansulfonic) acid, and 0.1% bovine serum
albumin at 37°C for periods that varied from 1–72 hours
depending on the experiment. The hormones were assayed in the media, blood, or
pituitary homogenates by radioimmunoassays. Surgical interventions like
castration from 1–46 days, chemical
treatments like in vitro exposure to testosterone, or pharmacological exposure
to drugs in vivo were routinely employed to manipulate the hormonal status.Figure 1
A photograph of a freshly dissected
pituitary-hypothalamus complex (PHC) from a normal adult male rat killed 5 minutes,
following intracardiac injection of India ink [24].


### 2.1. Hypothalamic Factors

Dutt et al. (1986) first used the adult rat PHc cultures as the method of choice to
study the inhibitory influence of hypothalamus over the kinetics of secretion
of prolactin from the adenohypophyseal lactotrophs in vitro [27]. The authors
observed that intact pituitary released comparable amounts of prolactin from 2
to 6 hours in vitro. The hourly basal rate of prolactin release decreased from
2 to 6 hours of incubation (Table 1). Coincubation with the hypothalamus
neither suppressed the amounts secreted nor altered the rate of pituitary
prolactin secretion from 2–6 hours. The data
was interpreted to suggest that the hypothalamic prolactin releasing factors
(PRFs) were necessary to maintain the basal secretion of prolactin from the
pituitary glands. The secretion of prolactin from PHc was observed to be
considerably lower than that from intact pituitary or coincubates. The rate of
prolactin release from PHc decreased from 4 to 6 hours. It could be inferred
from the comparative kinetics of prolactin secretion from intact pituitary and
PHc cultures that when the portal connections were maintained in vitro between
the adeno- and neurohypophyses and the neural and portal connections maintained
with the hypothalamus, the rate of prolactin secretion was more physiological
because of the availability of the hypothalamic PRFs and PIFs. However, the
functionality of the dopaminergic inhibitory receptors on the lactotrophs in
the PHc culture model only became evident when these systems were challenged
with a dose of 1×10−7 M dopamine in vitro. Only the PHc responded to
the dopamine challenge and exhibited a decrease in the rate of prolactin
secretion.Table 1
Release of Prolactin by different pituitary constructs in vitro.
SystemWhole pituitaryWhole pituitaryPituitary-hypothalamusPlus hypothalamuscomplexIncubation(h)TotalaPer hbTotalaPer hbTotalaPer hb2371.3±42.9185.6±21.5281.4±22.9140.7±11.5227.9±28.9b113.9±14.44360.2±34.690±8.7**294.9±31.173.7±7.8*345.4±21.9**86.4±5.56437.8±56.672.5±9.4*401.9±24.3**67±4.1*270.4±30.7c45±5.1*Pituitary constructs were incubated in DMEM at 37°C for 4 hours, in replicates of 10, at each time interval. Prolactin was
assayed in spent medium by RIA. Values are expressed as mean ± SEM. ang Prl (NIAMDD-RP-2); *P<.001, **P<.01, with respect to levels at 2 hours; bP<.02, cP<.05, with respect to corresponding pituitary
levels [27].Dopamine has been shown to inhibit the release of
prolactin in a variety of in vitro systems [28–32]. In another set of
experiments designed to further validate PHc as a model system of choice to
study prolactin release, Dutt et al. (1992) compared the effects of dopamine,
its agonists and antagonists in PHc, whole pituitary (PI), adenohypophysis
(P-N), P-N+PP (posterior pituitary) cultures from normal and reserpine-treated
(prolactin depleted) rats [33]. The authors observed that as compared to the
other systems, PHc, which released less prolactin than the other systems, also
responded adequately to agonists like 10−7 M dopamine, 10−7 M
Bromocriptine, 8×10−7 M Apomorphine with suppression, and
antagonists like 5×10−8 M Perphenazine with stimulation of
prolactin release, even after reserpine-induced depletion of lactotrophs (Table 2). The authors concluded that if the intact portal and neural connections
between the anterior and posterior lobes of the pituitary and the hypothalamus
were not maintained, the dopaminergic receptors on the lactotrophs no longer
responded to dopamine challenge in vitro, since the latter hormone, if not
stored in the posterior pituitary, got degraded rapidly [34]. It was inferred
from these studies that prolactin had a characteristic pattern of release in
vivo which was dependent on the delivery of hypothalamic PRFs and PIFs to the
neurohypophysis through neural connections and reached the adenohypophysis
through neurohypophyseal portal capillaries. Functional pituitary DA receptors
were absolutely essential for the dopaminergic inhibitory mechanism to operate,
both in vitro and in vivo, for maintaining homeostasis of prolactin.Table 2
Effects of reserpine on prolactin secretion by different pituitary constructs
in response to dopamine agonists and antagonists in vitro.
AdditionsControlReserpine 1.5 mg/kg/dIn 25% Ascorbic acidin
25% ascorbic acid(in vitro)PIP-NPHcPIP-NPHcAscorbic acid429 ± 14.8702 ± 8.54c316 ± 7.59126 ± 1.9b126 ± 3.8b167 ± 4.4bc(0.05%)Dopamine344 ± 12.34219 ± 11.39a124 ± 2.85a50 ± 1.27ab71 ± 3.8ab59 ± 1.9ab10−7 MBromocriptine317 ± 10.12339 ± 5.38a161 ± 5.06a46 ± 2.53ab56 ± 3.8ab50 ± 1.9ab10−7 MApomorphine364 ± 5.06108 ± 5.38a108 ± 11.71a40 ± 1.27ab60 ± 2.53ab30 ± 1.27b8×10−7 MPerphenazine393 ± 10.12694 ± 3.8c466 ± 5.7*140 ± 3.8140 ± 6.96330 ± 5.7*5×10−7 MReserpine was dissolved in 25% ascorbic acid and administered s.c to adult male rats for
46 hours. Control group rats received
vehicle alone. Different pituitary constructs were dissected out in DMEM and
incubated at 37°C for 4 hours, in replicates of 10, at each time interval.
Prolactin was assayed in spent medium by
RIA. Values are expressed as mean (ng NIDDK-RAT-PRL-RP-3-ml incubation medium) ± SEM. asignificant
difference with respect to 0.05% ascorbic acid (P≤.05). bsignificant
difference with respect to 25% ascorbic acid control group (P≤.05). csignificant difference with respect to PI and PHc of the same group (P≤.05). *significant difference with respect to PI and P-N of the same
group (P≤.05) [33].Having established the suitability of the PHc
cultures for studying the regulation of prolactin secretion in vitro, Karanth
et al. (1987) used the rat PHc and PI cultures to establish the ontogenetic
pattern of maturation of the hypothalamic releasing and release-inhibiting
activities that regulate its systemic levels at adulthood [35]. Several
endogenous factors have been reported to stimulate prolactin secretion viz.
Serotonin, β endorphin, Met enkephalin, Leu-enkephalin, thyrotropin-releasing
hormone (TRH), luteinizing hormone releasing hormone (LHRH), substance P,
vasopressin, oestradiol-17-β, epidermal growth factor, fibroblast growth
factor, cholecystokinin, and angiotensin II, prolactin-releasing peptides
(PrRPs) by direct action at the pituitary level [36–40]. To qualify as PRFs,
these factors would have to override the inhibitory control of prolactin
secretion by physiological PIFs like dopamine and GABA [41–43]. The prolactin
released by the intact pituitary in vitro is a measure of unregulated release
whereas that secreted by PHc in vitro is the cumulative effect of physiological
PRFs. Dutt et al. (1986) assumed that since PHc secreted less prolactin than
intact hypophysis in vitro, the difference in the amount released by the two
systems would reflect the cumulative hypothalamic PIF activity (HPIA) whereas
that secreted by PHc should be a true reflection of hypothalamic PRF activity
[27]. Karanth et al. (1987) used PHc and PI cultures to validate this
hypothesis. They attempted to correlate the developmental changes in the HPRA
and HPIA activities in terms of prolactin released from PHc and PI in vitro
from postnatal day 7 to 77, to the age-related changes in the serum prolactin
levels in male rats, as reported in literature [35, 44]. The pituitary weights
and prolactin content increased from day 7 to 70 but was maintained thereafter
in adult rats. The weight of the hypothalamus increased from postnatal day 7 to
56 before stabilization at maturity. Serum prolactin levels underwent
age-related increase from birth to 77 days with two peaks at days 49 and
77. The HPIA activity was undetectable
in vitro prior to day 21, peaked from days 35 to 49, decreased at day 70, and
was maintained thereafter. The HPRA activity increased in vitro through days 7
to 56, decreased on day 63, and stabilized (see Figure 2). The data was
suggestive of the presence of potent PRF activity prior to age 21. The
inference drawn from this study was supported by the findings of Becú &
Libertun (1982) and Khorram et al. (1984) who reported potent age-related PRF
activities viz. TRH activity on day 1 and serotonergic PRF activity on day 12
in male rats [45, 46]. Thus, the authors concurred that age-related increase in
serum prolactin levels till day 28 could be attributed to the concomitant
increase in HPRA. Since the serum prolactin levels peaked again at day 70,
despite stabilization of HPIA and HPRA activities, it was inferred that extrahypothalamic
factors like gonadal estradiol could be modulating the responsiveness of the
pituitary to dopamine. Alternatively, changes in the sensitivity of pituitary
to HPIA and HPRA could have led to increased secretion at adulthood. This
conclusion was based on the prevailing hypothesis that the responsiveness of
pituitary to TRH, LHRH, DA, and estradiol undergoes a change from puberty to
adulthood [47–50]. It was inferred that the levels of prolactin in circulation
were determined by competitive interactions between HPIA and HPRA, in
accordance with the suggestion of Matsushita et al. [51–53].Figure 2
Age-related changes in serum concentrations of
prolactin (dotted line), release of prolactin by whole pituitary (free release; dashed line),
hypothalamic prolactin-releasing activity (solid line), and hypothalamic prolactin-inhibiting activity (dotted-dashed line) in male rats.
Values are mean ± SEM from
Values are mean ± SEM from
two separate experiments and a minimum number of 14 animals per pooled group.*P<.05 compared with preceding group; †P<.05 compared with hypothalamic
prolactin-releasing activity at the same age (Student’s t-test). The inset shows the results for days 7 and 14 on an expanded
scale. The pituitary constructs were dissected
out of 7 to 77 day-old rats and incubated in DMEM for 4 hours at 37°C.
Prolactin was estimated by RIA in the spent medium [35].


### 2.2. Testicular Factors

Prolactin can upregulate the synthesis of testosterone in vivo through cognate receptors
on the Leydig cells of the testis [21, 22]. Based on intuitive logic that end
products are generally involved in the feedback regulation of their hormonal
stimulus, Gill-Sharma et al. (1992) studied the inhibitory effects, if any of
testosterone on prolactin secretion from rat PHc and intact pituitary of normal
and 7-day castrated rats in vitro [54]. The authors could demonstrate the
existence of a novel long loop feedback mechanism for the regulation of
prolactin release in sexually competent male rats. They observed that temporal
release of prolactin from PHc of normal rats was constant from 24 to 72 hours
whereas that from PHc from castrated and intact pituitary of normal and
castrated rats decreased from 24 to 72 hours of incubation in vitro (see Figure 3). The release rates per 24 hours from normal PHc constructs were significantly
lower than those from intact pituitary constructs. The decrease in intact
pituitary basal release rates could be attributed to absence of hypothalamic
PRFs. The decrease in release from castrated PHc and intact pituitary
constructs was likely due to either the lack of PRFs or availability of PIFs
from the median eminence or both. Coincubation with testosterone suppressed the
temporal secretion rate of prolactin from both PHc and intact pituitary
constructs of normal and castrated rats from 24 to 72 hours in vitro. This
observation was interpreted to suggest that testosterone primarily acted on the
pituitary lactotrophs of both constructs to suppress prolactin secretion.
Interestingly, prolactin content of incubated pituitaries from PHc constructs of
normal rats was lower than that in intact pituitaries but this difference
disappeared after castration. This observation was suggestive of suppressive effects of
testosterone on prolactin synthesis. The inhibitory effect of testosterone
became evident from the clear suppression of prolactin content both in
incubated pituitaries from PHc and intact pituitaries of normal and castrated
rats (see Figure 4). The functionality of the 72-hour incubated pituitary
constructs was demonstrable when all pituitary constructs not exposed to
testosterone, responded to a challenge dose of 100 pm LHRH over a 4-hour
period, with enhanced release of LH and FSH, whereas androgen exposure
counteracted this response. The underlying mechanism through which testosterone
suppressed prolactin remains to be delineated at the biochemical level. It can
be speculated that prolactin receptors expressed on the pituitary lactotrophs
could have mediated this effect [55]. Prolactin has been reported to exert a
proliferative effect on pituitary GH3 cells, expressing a cognate receptor
[56]. Hyperplasia of the adenohypophysis is a hallmark of mice with
dysfunctional prolactin signaling, akin to the effect observed in dopamine
receptor D2-deficient mice, and suggestive of an antiproliferative role of
hypothalamic dopamine in antagonizing the prolactin receptor-mediated growth
response [14, 57]. It remains to be seen whether testosterone can exert an
antiproliferative effect on lactotrophs by downregulating the prolactin
receptor, observed in rat liver, to eventually reduce the level of prolactin
[58].Figure 3
Release of PRL by PHC and PI of normal and castrated
rats incubated with or without testosterone for 72 hours. Upper panel. Pattern
of release of PRL by PHC and PI of castrated rats, incubated in absence (▴) or presence (▪) of testosterone (0.3 mM) for 72 hours. Values are mean ± SE and derived from 40 individual incubations
of PHC and 16 of PI, respectively. Lower panel. Pattern of release of PRL by
PHC and PI of normal rats incubated in absence (∘) or presence (•) of testosterone (0.3 mM) for 72 hours. Values are mean ± SE and derived from 15 individual incubations
of PHC and 16 PI, respectively. a, b, c denote significant difference in values at 48 hours
as compared to 24 hours, at 72 hours as compared to 48 hours, and at 72 hours
as compared to 24 hours, respectively. p, q, r denote significant difference in groups incubated
with or without testosterone at 24, 48, and 72 hours, respectively. *P≤.05. Pituitary constructs were dissected out of normal
or 7-day castrated rats and incubated in DMEM with or without 0.3 mM
testosterone, at 37°C for 24–72 hrs. Prolactin was analysed in the spent medium by RIA
[54].Figure 4
Intrapituitary contents of PRL after incubation of
PHC (left-hand panel)
and PI (right-hand panel) from normal rats without (stippled bars) or with (hatched bars) testosterone (0.3 mM) or from castrated rats
incubated without (open bars) or with (solid bars) testosterone (0.3 mM) for 72 hours. Values represent mean ± SE derived from 10 pituitaries.p, q, r, denote significant
differences in groups incubated with or without testosterone, normal controls versus
castrated controls and between normal incubated with testosterone versus castrated
incubated with testosterone, respectively. *P≤.05. C: Incubation without
testosterone; T: Incubation with testosterone. Pituitary prolactin content was
estimated by RIA in PBS homogenates of pituitary constructs incubated with or
without 0.3 mM testosterone for 72 hours and a challenge dose of LHRH at 37°C for
4 hours [54].Gill-Sharma et al. (2001) had reported that the
suppression of LH induced by low doses of estradiol in male rats did not
inhibit the secretion of testosterone presumably due to an effect of prolactin on
its synthesis in the Leydig cells [59]. Testosterone inhibition however
occurred at higher estradiol doses only after enhancement of systemic prolactin
levels (see Table 3). Presumably, estradiol-induced enhancement of rat
prolactin secretion in vivo occurs through estrogen response elements in its
gene in the pituitary gland and leads to infertility [60].Table 3
Effects of estradiol 17β on serum hormones in adult male rat.
Estradiol 17βPlasma
levels (ng/mL)(μg/kg/d)LHFSHPRLT00.85±0.096.16±0.32122.37±6.172.17±0.310.10.89±0.1085.58±0.498115.80±4.031.50±0.17100.54±0.109*3.49±0.38*120.60±4.701.44±0.191000.43±0.09*2.38±0.24*255.50±17.9*0.72±0.04*2000.31±0.08*2.67±0.17*306.00±22.27*0.84±0.09*3000.19±0.05*2.65±0.13*370.00±13.60*0.89±0.06*4000.17±0.03*2.48±0.56*364.30±38.07*0.83±0.10*10000.36±0.096*2.98±0.18*465.00±51.19*0.63±0.097*Estradiol
17β  was dissolved in saline by sonication and
injected s.c. Male rats were mated at 60 days of treatment and serum hormones
analyzed by RIA. All values are expressed as mean ± SEM. Dose “0” represents
control values. *denotes significance at P≤0.5 as compared to
controls [59].These studies had brought out the importance of
prolactin in maintaining testosterone homeostasis and fertility status of male
rats. Although the significance of testosterone to male fertility is well
documented, the studies of Aleem et al. (2008) demonstrated for the first time
the consequences of inadequate levels of this hormone to fertility in male rats
[61] see (Figure 5).
They demonstrated that blocking the androgen receptors with cyproterone
acetate (CPA), at a dose of 20 mg/Kg/d for 15 days, to reduce the
bioavailability of testosterone, led to underprotamination of epididymal sperm
chromatin during spermiogensis in sexually competent male rats (see
Figure 5). Our recent studies using CMA3 fluorescent dye uptake assay have
confirmed the earlier observations of Aleem et al. (2005) in male rats.
Cyproterone acetate-treated spermatozoa from caput epididymides incorporated
the CMA3 dye at the GC-rich regions lacking protamine whereas the control rat spermatozoa were unreactive
(Figures 6(a)–6(d))
[62].Figure 5
Western blots of protamine 1 in epididymal sperm of sexually mature rats depicting the
presence of 6 Kd protamine 1 bands in control and absence in drug-treated rats.
Cp: Cyproterone acetate; C: Control; N: negative control. Basic proteins were
extracted in HCl, differentially extracted in perchloric acid, analysed on 15%
acid urea PAGE. Western blotting was done with 1Hup 1N monoclonal antibody
provided by Dr. Rod Balhorn [61].Figure 6
(a) Spermatozoa
taken from untreated rats that have not picked up the CMA3 chromatin stain viewed under fluorescent
microscope at 100 X magnification at 500–610 nm excitation/fluorescence
emission wavelength. (b) Spermatozoa
of untreated rats viewed under phase contrast objective at 40 X magnification. (c)-(d) Spermatozoa taken from the caput epididymides of CPA-treated
rats that have picked up the intense yellow CMA3 nuclear chromatin stain in GC-rich regions normally occupied by
protamine. Spermatozoa were fixed in Carnoy’s fixative and stained with CMA3
dye in McIlvaine’s buffer. Staining was viewed under fluorescent microscope at
100 X magnification at 500–610 nm excitation/fluorescence emission wavelength.


### 2.3. Hypophyseal Factors

In vitro studies on PHc system further led to the
development of several in vivo models in our lab with a view to understanding
the physiological relevance of the long and short feedback mechanisms involved
in the regulation of pituitary hormones. Prolactin levels in the serum are
known to be autoregulated through prolactin receptors expressed on the
hypothalamic dopaminergic neurons [63]. The
autoregulation of prolactin, referred to as the short feedback regulation,
appears to be age-related and is purported to be mediated either through
retrograde flow in the hypophyseal stalk or via cerebrospinal fluid (CSF) as a
result of receptor-mediated uptake at the choroid plexuses [64, 65]. Balasinor
et al. (1992) carried out studies in long-term castrated male rats and
demonstrated that CSF is a vital link between the circulating prolactin and
hypothalamic dopaminergic neurons that mediate its autoregulation [66]. The
authors observed that bilateral castration of adult rats for
1, 3, 7, 14, 21, 28, 35, 46 days led to an increase in serum immunoactive prolactin
levels but those in the CSF, removed from cisterna magna, displayed an inverse
correlation (Figure 7). The turnover of hypothalamic dopamine decreased in
castrated rats suggestive of a breakdown in the feedback mechanism in the
dopaminergic neurons. Apparently either systemic bioactive prolactin was low or
castration had led to a breakdown of receptor-mediated uptake mechanism at the
choroid plexuses, as was suggested by low levels in CSF, with the result that
prolactin was not reaching the dopaminergic neurons. Alternatively,
dopaminergic neurons had become refractory to prolactin. It could be reasoned
that estradiol of adrenal origin could have gained access to the dopaminergic
neurons, downregulated dopamine turnover, and prevented prolactin
autoregulation. In confirmation, a recent study by Aleem et al. (2006) suggested
that administration of exogenous estradiol, at a dose of 100 ug/Kg/d, raised its
systemic levels in male rats twofold within 10 days, concomitant with a
fourfold increase in the secretion of prolactin [67]. Furthermore, Aleem et al.
(2005), also observed that when the antiandrogen cyproterone acetate was
administered to male rats at a dose of 20 mg/Kg/d for 15 days, a twofold
decrease in the serum estradiol levels produced a twofold suppression of
prolactin secretion [62].Comparative levels of PRL in the serum and CSF of
male rats at different days following castration. Upper panels. Serum PRL
levels. Lower panels. CSF PRL levels. Values are expressed in terms of
NIADDK-rat-PRL-RP-3. “c” denotes significant difference at P<.05 level with respect to
intact controls. Solid line with open circle (-∘-), dashed line with cross (–×–), and dotted line with closed
circle (⋯•⋯) represent data from
castrated, intact, and sham operated rats, respectively. At each day of
castration, values are expressed as mean ± SEM of three or more determinations in duplicate of
serum/CSF samples. In intact and sham-operated controls, each value is a mean
of two determinants in duplicate of serum/CSF. Small dots (•) denote value of each
determination. Vertical bars represent SEM. a, b denote levels of significant difference at P<.001 or P<.01 with respect to sham and intact controls. Prolactin was
estimated by RIA in serum and CSF of normal and 1–46 day castrated rats
[66].


## 2.1. Hypothalamic Factors

Dutt et al. (1986) first used the adult rat PHc cultures as the method of choice to
study the inhibitory influence of hypothalamus over the kinetics of secretion
of prolactin from the adenohypophyseal lactotrophs in vitro [27]. The authors
observed that intact pituitary released comparable amounts of prolactin from 2
to 6 hours in vitro. The hourly basal rate of prolactin release decreased from
2 to 6 hours of incubation (Table 1). Coincubation with the hypothalamus
neither suppressed the amounts secreted nor altered the rate of pituitary
prolactin secretion from 2–6 hours. The data
was interpreted to suggest that the hypothalamic prolactin releasing factors
(PRFs) were necessary to maintain the basal secretion of prolactin from the
pituitary glands. The secretion of prolactin from PHc was observed to be
considerably lower than that from intact pituitary or coincubates. The rate of
prolactin release from PHc decreased from 4 to 6 hours. It could be inferred
from the comparative kinetics of prolactin secretion from intact pituitary and
PHc cultures that when the portal connections were maintained in vitro between
the adeno- and neurohypophyses and the neural and portal connections maintained
with the hypothalamus, the rate of prolactin secretion was more physiological
because of the availability of the hypothalamic PRFs and PIFs. However, the
functionality of the dopaminergic inhibitory receptors on the lactotrophs in
the PHc culture model only became evident when these systems were challenged
with a dose of 1×10−7 M dopamine in vitro. Only the PHc responded to
the dopamine challenge and exhibited a decrease in the rate of prolactin
secretion.Table 1
Release of Prolactin by different pituitary constructs in vitro.
SystemWhole pituitaryWhole pituitaryPituitary-hypothalamusPlus hypothalamuscomplexIncubation(h)TotalaPer hbTotalaPer hbTotalaPer hb2371.3±42.9185.6±21.5281.4±22.9140.7±11.5227.9±28.9b113.9±14.44360.2±34.690±8.7**294.9±31.173.7±7.8*345.4±21.9**86.4±5.56437.8±56.672.5±9.4*401.9±24.3**67±4.1*270.4±30.7c45±5.1*Pituitary constructs were incubated in DMEM at 37°C for 4 hours, in replicates of 10, at each time interval. Prolactin was
assayed in spent medium by RIA. Values are expressed as mean ± SEM. ang Prl (NIAMDD-RP-2); *P<.001, **P<.01, with respect to levels at 2 hours; bP<.02, cP<.05, with respect to corresponding pituitary
levels [27].Dopamine has been shown to inhibit the release of
prolactin in a variety of in vitro systems [28–32]. In another set of
experiments designed to further validate PHc as a model system of choice to
study prolactin release, Dutt et al. (1992) compared the effects of dopamine,
its agonists and antagonists in PHc, whole pituitary (PI), adenohypophysis
(P-N), P-N+PP (posterior pituitary) cultures from normal and reserpine-treated
(prolactin depleted) rats [33]. The authors observed that as compared to the
other systems, PHc, which released less prolactin than the other systems, also
responded adequately to agonists like 10−7 M dopamine, 10−7 M
Bromocriptine, 8×10−7 M Apomorphine with suppression, and
antagonists like 5×10−8 M Perphenazine with stimulation of
prolactin release, even after reserpine-induced depletion of lactotrophs (Table 2). The authors concluded that if the intact portal and neural connections
between the anterior and posterior lobes of the pituitary and the hypothalamus
were not maintained, the dopaminergic receptors on the lactotrophs no longer
responded to dopamine challenge in vitro, since the latter hormone, if not
stored in the posterior pituitary, got degraded rapidly [34]. It was inferred
from these studies that prolactin had a characteristic pattern of release in
vivo which was dependent on the delivery of hypothalamic PRFs and PIFs to the
neurohypophysis through neural connections and reached the adenohypophysis
through neurohypophyseal portal capillaries. Functional pituitary DA receptors
were absolutely essential for the dopaminergic inhibitory mechanism to operate,
both in vitro and in vivo, for maintaining homeostasis of prolactin.Table 2
Effects of reserpine on prolactin secretion by different pituitary constructs
in response to dopamine agonists and antagonists in vitro.
AdditionsControlReserpine 1.5 mg/kg/dIn 25% Ascorbic acidin
25% ascorbic acid(in vitro)PIP-NPHcPIP-NPHcAscorbic acid429 ± 14.8702 ± 8.54c316 ± 7.59126 ± 1.9b126 ± 3.8b167 ± 4.4bc(0.05%)Dopamine344 ± 12.34219 ± 11.39a124 ± 2.85a50 ± 1.27ab71 ± 3.8ab59 ± 1.9ab10−7 MBromocriptine317 ± 10.12339 ± 5.38a161 ± 5.06a46 ± 2.53ab56 ± 3.8ab50 ± 1.9ab10−7 MApomorphine364 ± 5.06108 ± 5.38a108 ± 11.71a40 ± 1.27ab60 ± 2.53ab30 ± 1.27b8×10−7 MPerphenazine393 ± 10.12694 ± 3.8c466 ± 5.7*140 ± 3.8140 ± 6.96330 ± 5.7*5×10−7 MReserpine was dissolved in 25% ascorbic acid and administered s.c to adult male rats for
46 hours. Control group rats received
vehicle alone. Different pituitary constructs were dissected out in DMEM and
incubated at 37°C for 4 hours, in replicates of 10, at each time interval.
Prolactin was assayed in spent medium by
RIA. Values are expressed as mean (ng NIDDK-RAT-PRL-RP-3-ml incubation medium) ± SEM. asignificant
difference with respect to 0.05% ascorbic acid (P≤.05). bsignificant
difference with respect to 25% ascorbic acid control group (P≤.05). csignificant difference with respect to PI and PHc of the same group (P≤.05). *significant difference with respect to PI and P-N of the same
group (P≤.05) [33].Having established the suitability of the PHc
cultures for studying the regulation of prolactin secretion in vitro, Karanth
et al. (1987) used the rat PHc and PI cultures to establish the ontogenetic
pattern of maturation of the hypothalamic releasing and release-inhibiting
activities that regulate its systemic levels at adulthood [35]. Several
endogenous factors have been reported to stimulate prolactin secretion viz.
Serotonin, β endorphin, Met enkephalin, Leu-enkephalin, thyrotropin-releasing
hormone (TRH), luteinizing hormone releasing hormone (LHRH), substance P,
vasopressin, oestradiol-17-β, epidermal growth factor, fibroblast growth
factor, cholecystokinin, and angiotensin II, prolactin-releasing peptides
(PrRPs) by direct action at the pituitary level [36–40]. To qualify as PRFs,
these factors would have to override the inhibitory control of prolactin
secretion by physiological PIFs like dopamine and GABA [41–43]. The prolactin
released by the intact pituitary in vitro is a measure of unregulated release
whereas that secreted by PHc in vitro is the cumulative effect of physiological
PRFs. Dutt et al. (1986) assumed that since PHc secreted less prolactin than
intact hypophysis in vitro, the difference in the amount released by the two
systems would reflect the cumulative hypothalamic PIF activity (HPIA) whereas
that secreted by PHc should be a true reflection of hypothalamic PRF activity
[27]. Karanth et al. (1987) used PHc and PI cultures to validate this
hypothesis. They attempted to correlate the developmental changes in the HPRA
and HPIA activities in terms of prolactin released from PHc and PI in vitro
from postnatal day 7 to 77, to the age-related changes in the serum prolactin
levels in male rats, as reported in literature [35, 44]. The pituitary weights
and prolactin content increased from day 7 to 70 but was maintained thereafter
in adult rats. The weight of the hypothalamus increased from postnatal day 7 to
56 before stabilization at maturity. Serum prolactin levels underwent
age-related increase from birth to 77 days with two peaks at days 49 and
77. The HPIA activity was undetectable
in vitro prior to day 21, peaked from days 35 to 49, decreased at day 70, and
was maintained thereafter. The HPRA activity increased in vitro through days 7
to 56, decreased on day 63, and stabilized (see Figure 2). The data was
suggestive of the presence of potent PRF activity prior to age 21. The
inference drawn from this study was supported by the findings of Becú &
Libertun (1982) and Khorram et al. (1984) who reported potent age-related PRF
activities viz. TRH activity on day 1 and serotonergic PRF activity on day 12
in male rats [45, 46]. Thus, the authors concurred that age-related increase in
serum prolactin levels till day 28 could be attributed to the concomitant
increase in HPRA. Since the serum prolactin levels peaked again at day 70,
despite stabilization of HPIA and HPRA activities, it was inferred that extrahypothalamic
factors like gonadal estradiol could be modulating the responsiveness of the
pituitary to dopamine. Alternatively, changes in the sensitivity of pituitary
to HPIA and HPRA could have led to increased secretion at adulthood. This
conclusion was based on the prevailing hypothesis that the responsiveness of
pituitary to TRH, LHRH, DA, and estradiol undergoes a change from puberty to
adulthood [47–50]. It was inferred that the levels of prolactin in circulation
were determined by competitive interactions between HPIA and HPRA, in
accordance with the suggestion of Matsushita et al. [51–53].Figure 2
Age-related changes in serum concentrations of
prolactin (dotted line), release of prolactin by whole pituitary (free release; dashed line),
hypothalamic prolactin-releasing activity (solid line), and hypothalamic prolactin-inhibiting activity (dotted-dashed line) in male rats.
Values are mean ± SEM from
Values are mean ± SEM from
two separate experiments and a minimum number of 14 animals per pooled group.*P<.05 compared with preceding group; †P<.05 compared with hypothalamic
prolactin-releasing activity at the same age (Student’s t-test). The inset shows the results for days 7 and 14 on an expanded
scale. The pituitary constructs were dissected
out of 7 to 77 day-old rats and incubated in DMEM for 4 hours at 37°C.
Prolactin was estimated by RIA in the spent medium [35].


## 2.2. Testicular Factors

Prolactin can upregulate the synthesis of testosterone in vivo through cognate receptors
on the Leydig cells of the testis [21, 22]. Based on intuitive logic that end
products are generally involved in the feedback regulation of their hormonal
stimulus, Gill-Sharma et al. (1992) studied the inhibitory effects, if any of
testosterone on prolactin secretion from rat PHc and intact pituitary of normal
and 7-day castrated rats in vitro [54]. The authors could demonstrate the
existence of a novel long loop feedback mechanism for the regulation of
prolactin release in sexually competent male rats. They observed that temporal
release of prolactin from PHc of normal rats was constant from 24 to 72 hours
whereas that from PHc from castrated and intact pituitary of normal and
castrated rats decreased from 24 to 72 hours of incubation in vitro (see Figure 3). The release rates per 24 hours from normal PHc constructs were significantly
lower than those from intact pituitary constructs. The decrease in intact
pituitary basal release rates could be attributed to absence of hypothalamic
PRFs. The decrease in release from castrated PHc and intact pituitary
constructs was likely due to either the lack of PRFs or availability of PIFs
from the median eminence or both. Coincubation with testosterone suppressed the
temporal secretion rate of prolactin from both PHc and intact pituitary
constructs of normal and castrated rats from 24 to 72 hours in vitro. This
observation was interpreted to suggest that testosterone primarily acted on the
pituitary lactotrophs of both constructs to suppress prolactin secretion.
Interestingly, prolactin content of incubated pituitaries from PHc constructs of
normal rats was lower than that in intact pituitaries but this difference
disappeared after castration. This observation was suggestive of suppressive effects of
testosterone on prolactin synthesis. The inhibitory effect of testosterone
became evident from the clear suppression of prolactin content both in
incubated pituitaries from PHc and intact pituitaries of normal and castrated
rats (see Figure 4). The functionality of the 72-hour incubated pituitary
constructs was demonstrable when all pituitary constructs not exposed to
testosterone, responded to a challenge dose of 100 pm LHRH over a 4-hour
period, with enhanced release of LH and FSH, whereas androgen exposure
counteracted this response. The underlying mechanism through which testosterone
suppressed prolactin remains to be delineated at the biochemical level. It can
be speculated that prolactin receptors expressed on the pituitary lactotrophs
could have mediated this effect [55]. Prolactin has been reported to exert a
proliferative effect on pituitary GH3 cells, expressing a cognate receptor
[56]. Hyperplasia of the adenohypophysis is a hallmark of mice with
dysfunctional prolactin signaling, akin to the effect observed in dopamine
receptor D2-deficient mice, and suggestive of an antiproliferative role of
hypothalamic dopamine in antagonizing the prolactin receptor-mediated growth
response [14, 57]. It remains to be seen whether testosterone can exert an
antiproliferative effect on lactotrophs by downregulating the prolactin
receptor, observed in rat liver, to eventually reduce the level of prolactin
[58].Figure 3
Release of PRL by PHC and PI of normal and castrated
rats incubated with or without testosterone for 72 hours. Upper panel. Pattern
of release of PRL by PHC and PI of castrated rats, incubated in absence (▴) or presence (▪) of testosterone (0.3 mM) for 72 hours. Values are mean ± SE and derived from 40 individual incubations
of PHC and 16 of PI, respectively. Lower panel. Pattern of release of PRL by
PHC and PI of normal rats incubated in absence (∘) or presence (•) of testosterone (0.3 mM) for 72 hours. Values are mean ± SE and derived from 15 individual incubations
of PHC and 16 PI, respectively. a, b, c denote significant difference in values at 48 hours
as compared to 24 hours, at 72 hours as compared to 48 hours, and at 72 hours
as compared to 24 hours, respectively. p, q, r denote significant difference in groups incubated
with or without testosterone at 24, 48, and 72 hours, respectively. *P≤.05. Pituitary constructs were dissected out of normal
or 7-day castrated rats and incubated in DMEM with or without 0.3 mM
testosterone, at 37°C for 24–72 hrs. Prolactin was analysed in the spent medium by RIA
[54].Figure 4
Intrapituitary contents of PRL after incubation of
PHC (left-hand panel)
and PI (right-hand panel) from normal rats without (stippled bars) or with (hatched bars) testosterone (0.3 mM) or from castrated rats
incubated without (open bars) or with (solid bars) testosterone (0.3 mM) for 72 hours. Values represent mean ± SE derived from 10 pituitaries.p, q, r, denote significant
differences in groups incubated with or without testosterone, normal controls versus
castrated controls and between normal incubated with testosterone versus castrated
incubated with testosterone, respectively. *P≤.05. C: Incubation without
testosterone; T: Incubation with testosterone. Pituitary prolactin content was
estimated by RIA in PBS homogenates of pituitary constructs incubated with or
without 0.3 mM testosterone for 72 hours and a challenge dose of LHRH at 37°C for
4 hours [54].Gill-Sharma et al. (2001) had reported that the
suppression of LH induced by low doses of estradiol in male rats did not
inhibit the secretion of testosterone presumably due to an effect of prolactin on
its synthesis in the Leydig cells [59]. Testosterone inhibition however
occurred at higher estradiol doses only after enhancement of systemic prolactin
levels (see Table 3). Presumably, estradiol-induced enhancement of rat
prolactin secretion in vivo occurs through estrogen response elements in its
gene in the pituitary gland and leads to infertility [60].Table 3
Effects of estradiol 17β on serum hormones in adult male rat.
Estradiol 17βPlasma
levels (ng/mL)(μg/kg/d)LHFSHPRLT00.85±0.096.16±0.32122.37±6.172.17±0.310.10.89±0.1085.58±0.498115.80±4.031.50±0.17100.54±0.109*3.49±0.38*120.60±4.701.44±0.191000.43±0.09*2.38±0.24*255.50±17.9*0.72±0.04*2000.31±0.08*2.67±0.17*306.00±22.27*0.84±0.09*3000.19±0.05*2.65±0.13*370.00±13.60*0.89±0.06*4000.17±0.03*2.48±0.56*364.30±38.07*0.83±0.10*10000.36±0.096*2.98±0.18*465.00±51.19*0.63±0.097*Estradiol
17β  was dissolved in saline by sonication and
injected s.c. Male rats were mated at 60 days of treatment and serum hormones
analyzed by RIA. All values are expressed as mean ± SEM. Dose “0” represents
control values. *denotes significance at P≤0.5 as compared to
controls [59].These studies had brought out the importance of
prolactin in maintaining testosterone homeostasis and fertility status of male
rats. Although the significance of testosterone to male fertility is well
documented, the studies of Aleem et al. (2008) demonstrated for the first time
the consequences of inadequate levels of this hormone to fertility in male rats
[61] see (Figure 5).
They demonstrated that blocking the androgen receptors with cyproterone
acetate (CPA), at a dose of 20 mg/Kg/d for 15 days, to reduce the
bioavailability of testosterone, led to underprotamination of epididymal sperm
chromatin during spermiogensis in sexually competent male rats (see
Figure 5). Our recent studies using CMA3 fluorescent dye uptake assay have
confirmed the earlier observations of Aleem et al. (2005) in male rats.
Cyproterone acetate-treated spermatozoa from caput epididymides incorporated
the CMA3 dye at the GC-rich regions lacking protamine whereas the control rat spermatozoa were unreactive
(Figures 6(a)–6(d))
[62].Figure 5
Western blots of protamine 1 in epididymal sperm of sexually mature rats depicting the
presence of 6 Kd protamine 1 bands in control and absence in drug-treated rats.
Cp: Cyproterone acetate; C: Control; N: negative control. Basic proteins were
extracted in HCl, differentially extracted in perchloric acid, analysed on 15%
acid urea PAGE. Western blotting was done with 1Hup 1N monoclonal antibody
provided by Dr. Rod Balhorn [61].Figure 6
(a) Spermatozoa
taken from untreated rats that have not picked up the CMA3 chromatin stain viewed under fluorescent
microscope at 100 X magnification at 500–610 nm excitation/fluorescence
emission wavelength. (b) Spermatozoa
of untreated rats viewed under phase contrast objective at 40 X magnification. (c)-(d) Spermatozoa taken from the caput epididymides of CPA-treated
rats that have picked up the intense yellow CMA3 nuclear chromatin stain in GC-rich regions normally occupied by
protamine. Spermatozoa were fixed in Carnoy’s fixative and stained with CMA3
dye in McIlvaine’s buffer. Staining was viewed under fluorescent microscope at
100 X magnification at 500–610 nm excitation/fluorescence emission wavelength.


## 2.3. Hypophyseal Factors

In vitro studies on PHc system further led to the
development of several in vivo models in our lab with a view to understanding
the physiological relevance of the long and short feedback mechanisms involved
in the regulation of pituitary hormones. Prolactin levels in the serum are
known to be autoregulated through prolactin receptors expressed on the
hypothalamic dopaminergic neurons [63]. The
autoregulation of prolactin, referred to as the short feedback regulation,
appears to be age-related and is purported to be mediated either through
retrograde flow in the hypophyseal stalk or via cerebrospinal fluid (CSF) as a
result of receptor-mediated uptake at the choroid plexuses [64, 65]. Balasinor
et al. (1992) carried out studies in long-term castrated male rats and
demonstrated that CSF is a vital link between the circulating prolactin and
hypothalamic dopaminergic neurons that mediate its autoregulation [66]. The
authors observed that bilateral castration of adult rats for
1, 3, 7, 14, 21, 28, 35, 46 days led to an increase in serum immunoactive prolactin
levels but those in the CSF, removed from cisterna magna, displayed an inverse
correlation (Figure 7). The turnover of hypothalamic dopamine decreased in
castrated rats suggestive of a breakdown in the feedback mechanism in the
dopaminergic neurons. Apparently either systemic bioactive prolactin was low or
castration had led to a breakdown of receptor-mediated uptake mechanism at the
choroid plexuses, as was suggested by low levels in CSF, with the result that
prolactin was not reaching the dopaminergic neurons. Alternatively,
dopaminergic neurons had become refractory to prolactin. It could be reasoned
that estradiol of adrenal origin could have gained access to the dopaminergic
neurons, downregulated dopamine turnover, and prevented prolactin
autoregulation. In confirmation, a recent study by Aleem et al. (2006) suggested
that administration of exogenous estradiol, at a dose of 100 ug/Kg/d, raised its
systemic levels in male rats twofold within 10 days, concomitant with a
fourfold increase in the secretion of prolactin [67]. Furthermore, Aleem et al.
(2005), also observed that when the antiandrogen cyproterone acetate was
administered to male rats at a dose of 20 mg/Kg/d for 15 days, a twofold
decrease in the serum estradiol levels produced a twofold suppression of
prolactin secretion [62].Comparative levels of PRL in the serum and CSF of
male rats at different days following castration. Upper panels. Serum PRL
levels. Lower panels. CSF PRL levels. Values are expressed in terms of
NIADDK-rat-PRL-RP-3. “c” denotes significant difference at P<.05 level with respect to
intact controls. Solid line with open circle (-∘-), dashed line with cross (–×–), and dotted line with closed
circle (⋯•⋯) represent data from
castrated, intact, and sham operated rats, respectively. At each day of
castration, values are expressed as mean ± SEM of three or more determinations in duplicate of
serum/CSF samples. In intact and sham-operated controls, each value is a mean
of two determinants in duplicate of serum/CSF. Small dots (•) denote value of each
determination. Vertical bars represent SEM. a, b denote levels of significant difference at P<.001 or P<.01 with respect to sham and intact controls. Prolactin was
estimated by RIA in serum and CSF of normal and 1–46 day castrated rats
[66].


## 3. Feedback Loops and Implications to Fertility Regulation in Male Rat

These in vitro and in vivo studies have confirmed
that gonadal steroids, estradiol, and testosterone together constitute a long
loop feedback mechanism for the regulation of prolactin secretion at the
pituitary hypothalamic level. Whereas estradiol or long-term deprivation of
testosterone stimulate the secretion of prolactin at the hypothalamic level,
suggestive of a positive long feedback, testosterone-mediated suppression in
vitro, and effects of castration in vivo indicate a negative feedback role at
the pituitary.The gaps in the information on the long loop
feedback regulation of prolactin emerged from the studies carried out by
Gill-Sharma et al. (2003) with the antipsychotic fluphenazine, a long-acting D2
dopamine receptor blocker, administered to male rats at a dose of 1–3 mg/Kg/d
for 60 days to male rats [68]. The authors reported that fluphenazine competed
with dopamine to block pituitary D2 receptors, leading to moderate
hyperprolactinemia through a compensatory upregulation of hypothalamic tyrosine
hydroxylase. Moderate increase in circulating prolactin led to a significant
suppression of LH and FSH but not
testosterone (Table 4). Grattan et al. (2007) reported that
intracerebroventricular injection of prolactin in estrogen-primed,
ovariectomized mice, led to the suppression of systemic LH [69].
The authors demonstrated that both
estradiol and prolactin induced prolactin receptor in the gonadotropin
releasing hormone (GnRH) neurons derived from transgenic mice expressing Green Fluorescent Protein (GFP) under the control of
GnRH promoter (GnRH-GFP) and implicated it in LH suppression. More recently,
Anderson et al. (2008) have reported that chronic hyperprolactinemia induced by
dopamine antagonist, Sulpiride, suppressed LH pulse frequency in estradiol-primed,
ovariectomized rat [70]. The authors further observed that estradiol augments
the effects of Sulpiride-induced prolactin on the expression of prolactin
receptor and associated inhibitory signaling molecules viz. STAT-5, SOCS-1 and
3, and CIS in the hypothalamic GnRH neurons of estrogen-primed ovariectomized
rat. The authors suggested that prolactin inhibited the LH pulses in vivo since
prolactin-induced suppression of GnRH has already been reported in immortalized GT1 cell
lines derived from murine GnRH neurons [71]. Such an effect of
hyperprolactinemia on LH had also been reported by Bartke et al. (1977) [72].
The chromatin of fluphenazine-treated epididymal spermatozoa was susceptible to
denaturation in vitro. The fertilizing potential of the poor quality of
spermatozoa produced by the fluphenazine-treated rats was severely compromised
as indicated by reduction in litters sired. Sires displayed a tendency to mate
later than their untreated counterparts indicative of an effect on reproductive
behaviour which was considered to be due to direct effects of moderately high
levels of prolactin on central incertohypothalamic neurons (IHDA). The authors
averred that the expected inhibition of testosterone did not occur due to a
direct effect of prolactin on its synthesis in the Leydig cells [68, 73]. In
support of the earlier observations suggestive of feedback repression by
testosterone, the in vivo study suggested that prolactin could autoregulate its
own secretion by upregulating the synthesis of testosterone. It is tempting to
speculate, on the basis of the observations of Jeyaraj et al. (2005) and Aleem
et al. (2006) that prolactin could also be playing a compensatory role in
maintaining circulating testosterone levels after sequestration of its
circadian peak by estradiol-induced androgen binding protein in the Sertoli
cells of rat testis [67, 74]. Thus the autoregulatory role of prolactin appears
to be exerted both at the hypothalamic and testicular levels. It can be
hypothesized on the basis of these results that the effects of moderate
hyperprolactinemia on fertility can be reversed by autoregulation through the
long and short feedback loops.Table 4
Effects of fluphenazine decanoate on fertility parameters and serum hormones in adult male rat.
AffectedFluphenazine treatment
(mg/kg/d) for 60 daysparameters0123Prolactin, ng/ml18.99±1.8276.80±11.1*82.53±10.6*139.30±25.1*LH, ng/ml1.13±0.260.23±0.04*0.44±0.05*0.54±0.05*FSH, ng/ml7.72±0.265.43±0.44*6.08±0.31*6.00±0.22*Testosterone, ng/ml2.60±0.142.17±0.172.40±0.202.32±0.24Estradiol, ng/ml82.00±9.51114.2±21.370.61±8.1458.19±10.4Sperm count, million/cauda63.8±3.8954.3±4.1852.8±3.5359±6.68Potency, %100±0083.3±11.24100±0066.7±14.21*Fecundity, %100±0066.7±21.0983.3±16.6750±22.36Implantation sites, per rat11.3±1.166.7±1.675.1±1.54*5.4±1.69*Litters sired, per female10.3±1.176.4±1.64.8±1.49*5.7±1.73*Fertility index, per rat0.9±0.080.6±0.140.5±0.15*0.5±0.15*Fluphenazine was dissolved in sesame oil and injected s.c. Male rats were mated at 60 days
of treatment and serum hormones analyzed by RIA. Uterine horns of mated females were exposed to count
implantation sites, ovaries for corpora lutea. All values are expressed as mean ± SEM. Dose “0” represents control
values.*denotes significance at P≤.05 as compared to controls.
Potency = % mated females; fecundity = % male rats siring at least one viable
pup; fertility index = ratio of implantation sites to corpora lutea; litter size
= number of pups per female [68].However, further studies by Aleem at al. (2005) with
fluphenazine in male rats did not support the corrective hypothesis on
fertility. They observed that in spite of the compensatory feedback
upregulation of testosterone by prolactin, the FSH levels remained
significantly suppressed [72, 75]. The effects of FSH inhibition could
be observed on the mechanism of sperm chromatin condensation during
spermiogenesis in the testis. The authors observed that levels of several key
proteins involved in chromatin condensation viz. cyclic adenosyl monophosphate
regulatory element modulator (CREMι),
transition proteins, and protamine 1 were suppressed in the testis. The
epididymal spermatozoa of treated male rats lacked protamination and were
loosely packaged (see Figure 8). Recent in vitro studies carried out in our lab
used fluorescent CMA3 dye uptake assay to demonstrate the protamination status
of spermatozoa. It was observed that spermatozoa taken from the caput
epididymides of untreated male rats did not incorporate the CMA3 dye in the
chromatin (see Figures 9(a)-9(b)). The uptake of
intense yellow CMA3 stain in the GC-rich regions of fluphenazine-treated
epididymal sperm chromatin confirmed the absence of protamination in these
regions where protamine normally binds (see Figures 9(c)-9(d)). Thus, in spite of
operational long and short feedback mechanisms, moderate hyperprolactinemia was
observed to lead to production of poor quality spermatozoa.Figure 8
Western blots of protamine 1 in epididymal sperm of
sexually mature rats depicting the presence of 6 Kd protamine 1 bands in
control and absence in drug-treated rats. F: Fluphenazine; C: Control; N: negative
control. Basic proteins were extracted in HCl, differentially extracted in
trichloroacetic acid, analysed on 15% acid urea PAGE. Western blotting was done with
1Hup 1N monoclonal antibody provided by Dr Rod Balhorn [61].Figure 9
(a) Spermatozoa
taken from untreated rats that have not picked up the nuclear chromatin stain
viewed under fluorescent microscope at 100 X magnification at 500–610 nm
excitation/fluorescence emission wavelength. (b) Spermatozoa of untreated rats that have not picked up the stain viewed
under phase contrast objective at 100 X magnification. (c)-(d) Spermatozoa taken from the caput epididymides of fluphenazine-treated
rats that have picked up the intense yellow CMA3 nuclear chromatin stain in the
GC-rich regions occupied normally by protamine. Spermatozoa were fixed in
Carnoy’s fixative and stained with CMA3 dye in McIlvaine’s buffer. Staining was
viewed under fluorescent microscope at 100 X magnification at 500–610 nm
excitation/fluorescence emission wavelength.


## 4. Feedback Loops and Fertility Regulation  in Men

Although it is not always possible to extrapolate the
results of animal studies to humans, studies carried out in our lab by Juneja
et al. (1991) suggested that feedback regulation by gonadal steroids might be a
conserved phenomenon [76]. The authors determined the pattern of
circadian release of reproductive hormones from the hourly blood samples drawn
from normal young men (Table 5). Their studies suggested that the
physiological relationship apparent between prolactin and testosterone in male
rats was also reflected in human males. The authors observed an inverse
correlation between the acrophase of circadian release of bioactive prolactin
with nadir of LH and a direct correlation with that of the acrophase of
testosterone release in men. The temporal coupling of prolactin release between
20 and 23 hours followed by suppression of bioactive LH between 24 and 2 hours
and peak release of testosterone between 1 and 5 hours was suggestive of a
direct role for prolactin in entraining testosterone release essential for male
behaviour while LH release was being restrained at the level of hypothalamic
GnRH and opiatergic neurons and adenohypophyseal gonadotrophs by both the
hormones. Interestingly, peak release of testosterone appeared to have led to
suppression of prolactin between 9 and 11 hours concomitantly with that of
testosterone itself. Testosterone nadir coincided with a surge of bioactive LH
between 9 and 11 hours suggesting that the combined feedback inhibition by the
two hormones would have been released to allow autopriming of the GnRH
receptors on the gonadotrophs and synthesis of LH receptors on the Leydig cells.
The suppression of testosterone probably led to a surge in estradiol between 15
and 18 hours, presumably due to activation of P450 aromatase in the Leydig
cells. Estradiol was then observed to entrain the prolactin peak between 20 and
23 hours. That the prolactin peak is of pituitary and not extrapituitary origin
could be averred from the fact that only hypophyseal prolactin gene has
estrogen response elements in its promoter [60]. Their studies suggested that
the temporal coupling between prolactin and testosterone could signify a
functional relationship that may be playing an important role in the regulation
of human male fertility. Although differences have been reported between the
prolactinergic mechanisms between rat and human, particularly in terms of
extrapituitary sites of synthesis, which would argue against extrapolations,
this particular study led to the tentative conclusion that gonadal steroids,
testosterone, and estradiol could constitute a conserved physiological long
feedback mechanism between gonads and adenohypophysis which likely ensures the
circadian release of prolactin in systemic circulation, and maintains male
fertility and libido. This view is supported by the fact that both rat and
human hypophyseal prolactin genes share estrogen response elements in their
promoters [77].Table 5
Diurnal hormone variations in adult men.
Hormone/mLMeanCircadianAcrophase (h)Nadir (h)concentrationamplitude(range)(range)T, ng3.56±1.94*3.1±0.8*1–59–12E2, pg55.9±26.9*38±7.2*15–1824–2BLH, mIU55.4±6.25*14.4±2.2*9–1224–2ILH, mIU7.4±1.7*3±0.6*1–515–17BPRL, ng4.9±1.7*2.6±0.4*20–239–11IPRL, ng11.6±2.0*4.1±0.8*23–411–15Hormones in the hourly blood samples of healthy men were assayed by RIA. Hormone
concentration and circadian amplitudes are expressed per milliliter plasma.* =
SD. T = testosterone; ILH = immunoreactive LH (NIADDK-LER-907); BLH = bioactive LH (WHO 2nd IRP 78/549);
BPRL = bioactive PRL (NIAMDD-hPRL-I-6); IPRL = immunoactive PRL
(NIAMDD-hPRL-I-6); E2= 17-β-estradiol [76].


## 5. Conclusions

The results of our in vitro and in vivo studies
suggest that prolactin is a crucial hormone for the regulation of male
fertility with a defined ontogenetic pattern of development of the hypothalamic
releasing and inhibitory factors that determine its level in circulation at
adulthood. Adult hypothalami contain prolactin-responsive dopaminergic neurons
for autoregulation as well as stimulation of opiatergic neurons involved in the
inhibition of GnRH neurons. In addition to the hypothalamic regulatory factors,
adenohyphyseal lactotrophs at adulthood express a testosterone-responsive
inhibitory mechanism for prolactin synthesis and estrogen-dependent stimulatory
mechanisms for prolactin secretion, whereas Leydig cells in the testis express
a prolactin-dependent regulatory mechanism for testosterone synthesis. The
prolactin responsive mechanisms constitute the biological substrates for the
pituitary-hypothalamo-gonadal feedback system in the male mammals, which relies
on the gonadal steroids, testosterone and estradiol for long-loop, and
prolactin for short-loop feedback mechanisms. These mechanisms would operate to
contain the adverse effects of reproductive stress and ensure that serum levels
of prolactin remain within the physiological range, because even mild-to-moderate
hyperprolactinemia, if allowed to become chronic, affects the quality of mature
spermatozoa and their fertilizing potential. Hyperprolactinemia also affects
reproductive behaviour in spite of normal testosterone levels. Importantly,
whereas the effects of acute hyperprolactinemia appear to be mediated via
testosterone inhibition, those due to moderate hyperprolactinemia would be a
consequence of FSH deficits.



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*Source: 687259-2008-11-03.xml*