Prolactin and male fertility: the long and short feedback regulation

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.

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].

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].

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].

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 trichloroacetic 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.

Figure 7

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. Prolactin was estimated by RIA in serum and CSF of normal and 1–46 day castrated rats [66].

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.