SERUM FOLLICLE-STIMULATING HORMONE AND LUTEINIZING HORMONE LEVELS IN MALE RATS WITH EXPERIMENTALLY INDUCED DAMAGE OF THE GERMINAL EPITHELIUM L. DEBELJUK
Centro de
Investigaciones en Reproducción, Facultad de Medicina, Universidad de Buenos Aires, Buenos Aires, Argentina
(Received 22 August 1974) SUMMARY
depletion of germinal cells from the seminiferous tubules on serum follicle\x=req-\ stimulating (FSH) and luteinizing (LH) hormones was studied in young male rats. A single administration of 1-(N,N-diethylcarbamylmethyl)-2-4-dinitropyrrole (ORF 1616) seemed to damage primary spermatocytes and especially young spermatids. Whenever young spermatids (stages 1\p=n-\8) were absent or at least decreased in number, a significant increase
The effect of the
in
serum
FSH, and in
some cases serum
LH,
was
detected. The administration of another
drug, bis(dichloroacetyl) diamine (WIN 18446) for 21 consecutive days induced a depletion of primary spermatocytes in the seminiferous tubules, which was followed by a depletion of spermatids, due to the lack of spermatocytes to generate them. By the time the seminiferous tubules showed partial or total disappearance of young spermatids, serum FSH and in some cases also serum LH levels were significantly raised. It is concluded that the germinal epithelium of rat testes exerts some kind of control on serum gonadotrophin levels. Young spermatids seem to be involved in this regulation. INTRODUCTION
The possibility that the secretion of pituitary gonadotrophins could be in part controlled by the germinal epithelium of the testes has been investigated by several workers (McCullagh & Schaffenburgh, 1952: Johnsen, 1970). In man, it was recently reported that abnormalities of spermatogenesis were accompanied by an increase in levels of serum follicle-stimulating hormone (FSH) (Rosen & Weintraub, 1971; van Thiel, Sherins, Myers & De Vita, 1972). In the rat, lesion of the germinal epithelium by antispermatogenic agents was also associated with an increase in pituitary and serum gonadotrophins (Katsch & Duncan, 1968; Debeljuk, Arimura & Schally, 1973; Gomes, Hall, Jain & Boots, 1973). In a previous report, it was demonstrated that in rats with massive and prolonged depletion of the germinal epithelium, serum gonadotrophins were significantly raised (Debeljuk et al. 1973). Moreover, the alteration of the progression of spermatogenesis by damaging spermatogonia with busulfan, induced a significant increment in serum FSH and LH levels. This increase was particularly marked when immature spermatids were lacking in the seminiferous tubules. To investigate further the role of spermatocytes and spermatids in the control of gonadotrophin secretion, it was decided to correlate the damage of these cells, induced by l-(./V,A/-diethylcarbamylmethyl)-2,4-dinitropyrrole (ORF 1616) or bis (dichloroacetyl) diamine (WIN 18446), with * Member of the Career of the Investigator, Consejo Nacional de Investigaciones Científicas y Técnicas, Argentina.
the
serum gonadotrophin levels. ORF 1616 and spermatids when administered to rats (Patanelli
WIN 18446 damage spermatocytes and & Nelson, 1964; Drobeck & Coulston,
1962).
MATERIALS AND METHODS
Adult male rats of the Wistar strain were used. The animals were housed in quarters equip¬ ped with controlled temperature (21 °C) and illumination (14 h light: 10 h darkness) and were fed Purina laboratory chow, fresh vegetables and water ad libitum. In the first part of the experiment, 50 rats were treated with ORF 1616 at a dosage of 200 mg/kg body weight, administered through a gastric tube as a single dose. ORF 1616 was finely suspended in a 1 % solution of tragacanth gum. Fifty more rats were similarly treated with the vehicle alone and served as controls. Groups of ten control and ten experimental rats were killed 3, 7, 14, 21, and 28 days after the administration of ORF 1616 or the vehicle alone. Blood was collected from the trunk, sera being collected after centrifugation and kept frozen until assayed for gonadotrophins. Testes, seminal vesicles and ventral prostates were removed, dissected free and weighed. Testes were then fixed in Bouin's fluid and processed for histological studies, periodic acid-Schiff's-haematoxylin was the stain used. One hundred tubules from equatorial sections of the testis from each animal within every group were examined. The absence or presence of a given cell type in each seminiferous tubule was recorded and expressed as the mean percentage for each group, with its respective standard error. In the second part of the experiment, 60 rats were treated with WIN 18446 at a daily dosage of 125 mg/kg body weight for 21 consecutive days, administered through a gastric tube. WIN 18446 was finely suspended in a 1 % solution of tragacanth gum. Sixty additional rats were similarly treated with the vehicle alone and served as controls. Groups of ten con¬ trol and ten experimental rats were killed by decapitation at intervals of 10, 15, 22, 29, 36, and 43 days after starting the treatment. Blood was collected from the trunk, sera were separated and kept frozen until assayed for gonadotrophins. Anterior pituitaries, testes, seminal vesicles and ventral prostates were dissected free and weighed. Testes were then processed for histological studies using the same methodology as described above. In all the cases, serum FSH was determined using the double-antibody radioimmunoassay described by Daane & Parlow (1971). Purified rat FSH was used for iodination with 12SI and NIAMDD-Rat FSH-RP1 was used as a standard preparation. Serum LH was determined following the double-antibody radioimmunoassay described by Niswender, Midgley, Monroe & Reichert (1968). Purified ovine LH (gift of Dr L. E. Reichert) was iodinated with 125I and NIH-LH-S17 was used as a standard preparation. This preparation gave a parallel dose-response curve compared with NIAMDD-Rat LH-RP1. The significance of the differences between control and experimental groups was tested by means of Student's r-test. RESULTS
Organ weights
ORF 1616
The final body weight was decreased in experimental animals only in the groups killed 3 days after treatment. At all intervals testicular weights were significantly lower in experi¬ mental rats as compared with control rats (Table 1). No changes were noted in seminal vesicle or ventral prostate weights at any of the times studied.
Histological findings Three days after the administration of ORF 1616, 12± 1-5 (s.e.m.) % of the seminiferous tubules lacked primary spermatocytes; 45±7-8% of the tubules contained abnormal spermatids, especially those in Golgi, cap and early acrosome phase, corresponding to step 1
Table 1.
with ORF 1616 on the organ rats (means ± s.e.m.) adult male young
Effects of treatment
Seminal
Time after treatment
Group
(days)
Control ORF 1616 Control
3 3 7 7 14 14
ORF 1616
Control ORF 1616 Control ORF 1616 Control ORF 1616 Ten rats per group
Testicular wt
Body wt (g) 2730+3-6 249-0+8-2* 2820+3-6 2720 + 9-1 3050 + 7-6
2940+8-2
21 21
3210 + 6-2
28 28
3050+15-3 3130 + 7-1
were
3070+6-8
used.
weights of
*
Centro de
Investigaciones en Reproducción, Facultad de Medicina, Universidad de Buenos Aires, Buenos Aires, Argentina
(Received 22 August 1974) SUMMARY
depletion of germinal cells from the seminiferous tubules on serum follicle\x=req-\ stimulating (FSH) and luteinizing (LH) hormones was studied in young male rats. A single administration of 1-(N,N-diethylcarbamylmethyl)-2-4-dinitropyrrole (ORF 1616) seemed to damage primary spermatocytes and especially young spermatids. Whenever young spermatids (stages 1\p=n-\8) were absent or at least decreased in number, a significant increase
The effect of the
in
serum
FSH, and in
some cases serum
LH,
was
detected. The administration of another
drug, bis(dichloroacetyl) diamine (WIN 18446) for 21 consecutive days induced a depletion of primary spermatocytes in the seminiferous tubules, which was followed by a depletion of spermatids, due to the lack of spermatocytes to generate them. By the time the seminiferous tubules showed partial or total disappearance of young spermatids, serum FSH and in some cases also serum LH levels were significantly raised. It is concluded that the germinal epithelium of rat testes exerts some kind of control on serum gonadotrophin levels. Young spermatids seem to be involved in this regulation. INTRODUCTION
The possibility that the secretion of pituitary gonadotrophins could be in part controlled by the germinal epithelium of the testes has been investigated by several workers (McCullagh & Schaffenburgh, 1952: Johnsen, 1970). In man, it was recently reported that abnormalities of spermatogenesis were accompanied by an increase in levels of serum follicle-stimulating hormone (FSH) (Rosen & Weintraub, 1971; van Thiel, Sherins, Myers & De Vita, 1972). In the rat, lesion of the germinal epithelium by antispermatogenic agents was also associated with an increase in pituitary and serum gonadotrophins (Katsch & Duncan, 1968; Debeljuk, Arimura & Schally, 1973; Gomes, Hall, Jain & Boots, 1973). In a previous report, it was demonstrated that in rats with massive and prolonged depletion of the germinal epithelium, serum gonadotrophins were significantly raised (Debeljuk et al. 1973). Moreover, the alteration of the progression of spermatogenesis by damaging spermatogonia with busulfan, induced a significant increment in serum FSH and LH levels. This increase was particularly marked when immature spermatids were lacking in the seminiferous tubules. To investigate further the role of spermatocytes and spermatids in the control of gonadotrophin secretion, it was decided to correlate the damage of these cells, induced by l-(./V,A/-diethylcarbamylmethyl)-2,4-dinitropyrrole (ORF 1616) or bis (dichloroacetyl) diamine (WIN 18446), with * Member of the Career of the Investigator, Consejo Nacional de Investigaciones Científicas y Técnicas, Argentina.
the
serum gonadotrophin levels. ORF 1616 and spermatids when administered to rats (Patanelli
WIN 18446 damage spermatocytes and & Nelson, 1964; Drobeck & Coulston,
1962).
MATERIALS AND METHODS
Adult male rats of the Wistar strain were used. The animals were housed in quarters equip¬ ped with controlled temperature (21 °C) and illumination (14 h light: 10 h darkness) and were fed Purina laboratory chow, fresh vegetables and water ad libitum. In the first part of the experiment, 50 rats were treated with ORF 1616 at a dosage of 200 mg/kg body weight, administered through a gastric tube as a single dose. ORF 1616 was finely suspended in a 1 % solution of tragacanth gum. Fifty more rats were similarly treated with the vehicle alone and served as controls. Groups of ten control and ten experimental rats were killed 3, 7, 14, 21, and 28 days after the administration of ORF 1616 or the vehicle alone. Blood was collected from the trunk, sera being collected after centrifugation and kept frozen until assayed for gonadotrophins. Testes, seminal vesicles and ventral prostates were removed, dissected free and weighed. Testes were then fixed in Bouin's fluid and processed for histological studies, periodic acid-Schiff's-haematoxylin was the stain used. One hundred tubules from equatorial sections of the testis from each animal within every group were examined. The absence or presence of a given cell type in each seminiferous tubule was recorded and expressed as the mean percentage for each group, with its respective standard error. In the second part of the experiment, 60 rats were treated with WIN 18446 at a daily dosage of 125 mg/kg body weight for 21 consecutive days, administered through a gastric tube. WIN 18446 was finely suspended in a 1 % solution of tragacanth gum. Sixty additional rats were similarly treated with the vehicle alone and served as controls. Groups of ten con¬ trol and ten experimental rats were killed by decapitation at intervals of 10, 15, 22, 29, 36, and 43 days after starting the treatment. Blood was collected from the trunk, sera were separated and kept frozen until assayed for gonadotrophins. Anterior pituitaries, testes, seminal vesicles and ventral prostates were dissected free and weighed. Testes were then processed for histological studies using the same methodology as described above. In all the cases, serum FSH was determined using the double-antibody radioimmunoassay described by Daane & Parlow (1971). Purified rat FSH was used for iodination with 12SI and NIAMDD-Rat FSH-RP1 was used as a standard preparation. Serum LH was determined following the double-antibody radioimmunoassay described by Niswender, Midgley, Monroe & Reichert (1968). Purified ovine LH (gift of Dr L. E. Reichert) was iodinated with 125I and NIH-LH-S17 was used as a standard preparation. This preparation gave a parallel dose-response curve compared with NIAMDD-Rat LH-RP1. The significance of the differences between control and experimental groups was tested by means of Student's r-test. RESULTS
Organ weights
ORF 1616
The final body weight was decreased in experimental animals only in the groups killed 3 days after treatment. At all intervals testicular weights were significantly lower in experi¬ mental rats as compared with control rats (Table 1). No changes were noted in seminal vesicle or ventral prostate weights at any of the times studied.
Histological findings Three days after the administration of ORF 1616, 12± 1-5 (s.e.m.) % of the seminiferous tubules lacked primary spermatocytes; 45±7-8% of the tubules contained abnormal spermatids, especially those in Golgi, cap and early acrosome phase, corresponding to step 1
Table 1.
with ORF 1616 on the organ rats (means ± s.e.m.) adult male young
Effects of treatment
Seminal
Time after treatment
Group
(days)
Control ORF 1616 Control
3 3 7 7 14 14
ORF 1616
Control ORF 1616 Control ORF 1616 Control ORF 1616 Ten rats per group
Testicular wt
Body wt (g) 2730+3-6 249-0+8-2* 2820+3-6 2720 + 9-1 3050 + 7-6
2940+8-2
21 21
3210 + 6-2
28 28
3050+15-3 3130 + 7-1
were
3070+6-8
used.
weights of
*
