Key words
prolactin - prolactin receptor - spermatogenesis - testis - male reproduction
Introduction
Prolactin (PRL) is a polypeptide hormone, which is primarily secreted by lactotropic
cells of the pituitary [1]
[2]. Juggling between varied functions, prolactin is known to have more actions than
all the other pituitary hormones together. It has multifaceted roles like osmoregulation,
growth and development, endocrine functions, metabolism, neurobiological functions,
immunomodulation as well as in reproduction. Believed to be an important factor in
female reproductive physiology, the role of this hormone has been intensively studied
in females and is involved in lactation, mammary gland development, maintenance of
corpus luteum, and maternal behavior [1]
[2]. Prolactin receptors are also present in male reproductive organs, the precise role
or any specific target organ of this hormone in male reproductive physiology remains
unknown. Lately, several animal and human studies have shown that prolactin positively
modulates various aspects in testicular function hinting at a crucial role in male
reproduction.
The release of prolactin is regulated by a wide variety of factors secreted from the
Hypothalamus-Pituitary-Gonadal axis (HPG axis). The systemic levels of prolactin depends
on the actions and effects of these factors which can be inhibitory or stimulatory
in nature. Classically, prolactin and dopamine are known to be antagonistic in nature.
Prolactin regulates its own release via a short feedback mechanism by acting on prolactin
receptors on hypothalamic dopaminergic neurons. This causes the release of dopamine
which has an inhibitory effect on lactotrophs via its cognate D2 receptor leading
to prolactin suppression. This demonstrates that dopamine has a crucial role in regulating
prolactin levels [3]
[4]. Prolactin is believed to control the gonadal function indirectly by regulating
the release of gonadotropins from the pituitary or by directly increasing the concentration
of LH receptors on Leydig cells in the testes. All these effects show that prolactin
works at neuroendocrine level and has a pivotal role in male reproduction. This review
summaries the existing evidences and work done on prolactin in males and tries to
put light on the actions of prolactin in male reproduction.
Prolactin and Receptor Localization in Testis
Prolactin and Receptor Localization in Testis
Prolactin
Prolactin is primarily secreted by the anterior lobe of pituitary, but there are extra-pituitary
sites such as placenta, spleen, skin, and lachrymal gland [5]
[6]. The most abundant form of prolactin found is of 23 kDa, although multiple variants
have been identified in mammals. These variants are a result of alternative splicing
and post-translational modifications such as glycosylation, phosphorylation, dimerization,
and polymerization [2]
[7].
Testis comprises of 2 cell types; somatic cells (Sertoli, Leydig and pertitubular
myoid cells) and germ cells (spermatogonia, spermatocytes, round, elongating, and
elongated spermatids). Spermatogenesis is a process, which is regulated by cross-talk
between the 2 cell types. It can be divided into 4 distinct phases; mitosis (proliferation
of spermatogonia), meiosis (reductional division from primary spermatocyte to round
spermatids), spermiogenesis (cytodifferentiation of round spermatids to spermatozoa),
and spermiation (release of spermatozoa into the lumen) [8]. Prolactin regulates various testicular functions by modulating the FSH and LH receptors
on Sertoli and Leydig cells, respectively. It is also involved in meiotic division
in germ cells [9]
[10]
[11]
[12]. By immunohistochemistry Roux et al. demonstrated the presence of prolactin in interstitial
cells, Sertoli cells and germ cells [13]. However, it was unclear if prolactin was produced, secreted or simply stored in
the testes. Another study carried out, observed the transcript and protein levels
of PRL in testis. PRL transcript was mainly found in Leydig cells, spermatogonia,
pachytene and preleptotene spermatocytes in both prepubertal and adult mice [14]. Guillaumot and Benahmed showed the presence of PRL transcript in the Sertoli cell
isolated in vitro after FSH treatment [15]. It was also shown that the levels in testis were lower as compared to the pituitary
levels. Using immunoblotting and immunofluorescence, protein levels were detected
in testis as well as sperm [14].
Prolactin receptor
Prolactin acts via a transmembrane receptor, PRL-R belonging to class 1 cytokine receptor
superfamily. This family includes receptor for various interleukins, leptin, erythropoietin
etc. Prolactin receptor is closely related to growth hormone receptor (GHR) sharing
about 30% sequence identity as well as sharing structural and functional features.
Although all these receptor are genetically unique, they have consensus amino acid
sequences in intracellular as well as extracellular domains which are highly conserved
in all of them. The receptor has multiple isoforms and different isoforms have been
identified in different tissues in different species [16]
[17].
One of the reason why the role of prolactin is difficult to fathom in male reproduction
is because there is a lot of contradiction in the data available. Wahlstrom et al.
demonstrated the presence of prolactin receptor in rat testes, however, negative staining
was observed in human testes [18]. Contradictorily, the presence of prolactin receptor was shown in germ cells of
human testes mainly in pachytene spermatocytes, secondary spermatocytes, round spermatids
(RS), and elongating spermatids. Spermatogonia, leptotene, zygotene, diplotene spermatocytes,
and elongated spermatids along with Sertoli cells were devoid of prolactin receptors.
Weak signal was observed in Leydig cells [19]. Hondo et al. have observed prolactin receptor transcripts in all spermatogenic
cells by in situ hybridization except in RS [20] ([Fig. 1]).
Fig. 1 Diagrammatic representation illustrating the presence of prolactin receptor in the
testes. Red arrow indicates transcript and blue arrow indicates protein localization.
Signalling pathway
Prolactin receptor has 2 distinct domains; extracellular and intracellular domain.
The extracellular domain has a ligand binding site to which prolactin binds and bring
about dimerization of the receptor. Intracellular domain is responsible for initiating
the signalling cascade for prolactin receptor. This occurs via transphosphorylation
of Janus kinase (Jak2), which is constitutively present on the intracellular domain
of the receptor. Jak2 brings about phosphorylation of other tyrosine residues on the
receptor. Phosphorylated tyrosine residues acts as binding site for transducer molecules
like signal transducer and activator of transcription (STATs) which binds via SH2
domain. Different types of STATs, mainly STAT (1, 3, 5a, 5b) are involved in activation
of downstream targets. Prolactin receptor also activates Ras/Raf/MAPK (mitogen-activated
protein kinase) pathway by stimulating a wide variety of transcription factors or
early genes by phosphorylation leading to various genomic effects which has involvement
in various biological functions [1]
[2].
Rodent Models to Study Prolactin and Its Receptor Functions
Rodent Models to Study Prolactin and Its Receptor Functions
PRL agonists and antagonists
The significance of prolactin and its receptor in male reproduction remains debatable
since very few studies have characterized the effect of over or under expression of
the same. Nag et al. studied the effect of prolactin suppression by administering
dopamine receptor agonist, 2-bromo-α-ergocriptine to adult male rats [12]. They observed significant reduction in reproductive organ weights and serum prolactin
level. Whereas serum LH levels were increased and FSH remained unaffected. Upon exogenous
treatment of prolactin to PRL deficient male rats resulted in the restoration of the
effects observed. Enumeration of spermatogenic cells from stage VII of seminiferous
epithelium showed significant reduction in the RS population, however the other spermatogenic
cells were unaffected. The RS count was restored after prolactin supplementation [12]. Another study carried out by Bartke in hypophysectomized mice has also shown the
role of prolactin during spermatogenesis. The study showed significant reduction in
RS population in hypophysectomized hereditary dwarf mice and upon prolactin treatment
observed an increase in the same [21]. These evidences highlights the potential role of prolactin in meiosis. Also studies
in our lab have shown that upon estrogen treatment to adult male rats, higher serum
levels of prolactin were observed [22]
[23]. Further, upon estrogen receptor (ERα and ERβ) specific ligands treatment also showed
high prolactin levels followed defects in spermatogenesis process. ERα agonist was
responsible for affecting spermiogenesis whereas ERβ was involved affecting spermiation
[24]
[25]. Chromatin Immunoprecipitation (ChIP) Sequencing for estrogen receptor ERα and ERβ
in germ cells of adult male rats showed significant enrichment of genes involved in
prolactin pathway suggesting a plausible role of prolactin in spermatogenesis (Unpublished
observations). However, further studies are on-going to validate our findings.
Gill-Sharma et al. studied the effect of fluphenazine, a known dopamine receptor antagonist
which causes hyperprolactinemia in adult male rats [26]. No effects were seen in the reproductive organ weights, however, serum LH and FSH
levels were reduced. No effects were observed in serum testosterone and estradiol
levels. The fertility studies showed significant reduction in potency, implantation
size and litter size. Further studies showed reduction in spermiogenesis related proteins
like transition proteins, protamine, CREMt, ABP protein levels in the testes. These
results suggests that hyperprolactinemia causes reduction in FSH levels without any
change in testosterone levels. The reduction in FSH resulted in lower levels of cAMP
which may have led to dysregulation of downstream spermiogenesis related genes which
are involved in chromatin condensation [27]. However, this study did not quantitate the RS population therefore it cannot be
speculated if the reduction in protein levels is due to true reduction in RS population
or not. In addition, direct effects of hyperprolactinemia independent of FSH effect
cannot be ruled out as prolactin receptors are present in testicular cells. Further
studies are required to validate the findings. Hyper- or hypo-prolactinemia effects
on gonadotropins can be possibly due to secondary changes brought about by prolactin
on hypothalamus and not due to effect on the pituitary. Hyperprolactinemia in rats
lead to decrease in LH levels. However, prolactin is known to increase LH receptors
and its responsiveness in the testes [9]. This explains why the plasma testosterone levels were not affected in hyperprolactinemia
despite decrease in LH levels in rodents.
Prolactin and its receptor knockouts
Prolactin and its receptor knockouts
Steger et al. developed PRL KO male mice to study their neuroendocrine and reproductive
functions. Compared to wild type males the PRL KO males showed reduction in dopamine,
LH, and FSH levels. However, no effect was seen on testosterone levels and fertility
parameters [28]. This study corroborates with the findings observed by Bartke in hypophysectomized
hereditary dwarf mice [21].
Another way to study prolactin function was to knockout its receptor. Binart et al.
carried out disruption of prolactin receptor gene (PRLR KO) in mice. No effects were
observed on body weights, reproductive organ weights, fertility parameters, hormonal
profile, testicular morphology, and sperm parameters. Overall no changes were observed
in the absence of prolactin and its receptor in reproductive functions [29]. It would be interesting to study the double knockouts for both prolactin and its
receptor to get a clearer picture of the exact role in neuroendocrine and reproductive
functions. Surprisingly, an effect is observed on the reproductive function when prolactin
is knockdown [12] but not when it is completely knocked out [29]. This suggests that abnormal level of prolactin affects the reproductive function
but there may be some alternate mechanism, for example, growth hormone pathway, to
compensate in its absence [14].
Human studies (hyper/hypoprolactinemia and its effects)
Human studies (hyper/hypoprolactinemia and its effects)
In the past few years several studies have shown the relation between hypo- and hyper-prolactinemia
and male reproductive behavior in humans. Hyperprolactinemia can be idiopathic, tumor
or drug induced and is one of the causes leading to altered sexual behavior like erectile
dysfunction (ED) and loss of libido [30]. It is also speculated that it can cause endocrine disturbances leading to abnormal
levels of gonadotropins and testosterone. Buvat et al. showed that 88% of males who
are hyperprolactemic had erectile dysfunction [31]. Carani et al. did not observe any symptoms of ED in hyperprolactemic males as compared
to control individuals [32]. However, this discrepancy could be due to small sample size. When screened for
hyperprolactinemia in ED patients, very few percent of them were found to be hyperprolactemic.
Boucher et al. studied the effects of bromocriptine in individuals with hyperprolactinemia
and demonstrated that the prolactin secretion was lowered and spermatogenesis which
was altered was restored back to normal [33]. Another study by Merino et al. showed high levels of prolactin in patients with
different infertile conditions like oligozoospermia, asthenozoospermia and azoospermia
[34]. However upon treatment with prolactin antagonist, bromocriptine the serum levels
of prolactin remained unaltered. These suggest that there are not sufficient evidences
to prove if hyperprolactinemia leads to ED. The role of bromocriptine as a prolactin
antagonist is well established and is used in treatment of ED. Colao et al. studied
the effect of dopamine agonist, quanagolide in patients with macroprolactinoma and
microprolactinoma which showed high levels of prolactin and lower testosterone levels
along with low sperm count and sperm motility [35]. However, after treatment, the prolactin levels were lowered and the other parameters
were return to basal levels. Similar results were obtained after treatment with cabergoline
which is also a dopamine agonist [36]
[37].
On the other extreme, very few studies have shown the association between hypoprolactinemia
and sexual dysfunction. Corona et al. demonstrated that individuals with sexual dysfunction
showed lower levels of prolactin and higher risk of ED and premature ejaculation [38]. Low prolactin levels suggests that there could be an increase in the dopamine levels
or could be due to altered serotonin pathway. Gonzales et al. showed a strong relationship
with hypoprolactinemia and diminished sperm motility. However no effects were observed
on basic sperm parameters [39].
Concluding Remarks
Prolactin is involved in a number of different biological functions including reproduction
[1]
[2]. The role of prolactin has been majorly explored in female reproduction, however,
there is a paucity of information of its involvement in male reproduction. Current
rodent models for prolactin and its receptor knockouts point at the fact that there
are a lot of ambiguities in reproductive parameters in males suggesting that the mechanism
by which prolactin and its receptor acts on spermatogenesis still remains blurred.
Till date, the strategies used by the researchers include knocking out either prolactin
or its receptor [28]
[29]. The major disadvantage of using these models is that, the absence of prolactin
can be compensated by other molecules such as hormones or cytokines, also, in the
absence of prolactin receptor the different isoforms of prolactin can act via different
homologous receptor [14]. Using the current models to study the exact role of prolactin is difficult due
to its compensatory nature and therefore a better strategy is required to study prolactin,
for example, in the development of double knockout rodent models (Prl+Prlr) ([Fig. 2]). This will further expand our understanding of functioning of this multitasking
hormone.
Fig. 2 Schematic representation of different rodent models, which are deficient in prolactin
and its receptor.
Funding
This work [REV/668/08–2018] did not receive any specific grant from any funding agency
in the public or not-for-profit sector.
