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ABSTRACT

SEXUAL BEHAVIOR IN MALE OFFSPRING
OF FEMALE DIABETIC RATS

BY

Patricia Hilley Ruppert

Diabetes mellitus currently afflicts an estimated
5 million Americans. Despite treatment with insulin and
oral antidiabetic agents, many of the concomitants of
diabetes remain problematic. Clinical studies have shown
that 50 percent of diabetic males are impotent, although
the etiology of this disturbance remains undetermined.
Also, despite better prenatal and postnatal care, diabetic
pregnancy continues to pose a greater risk for mother and
offspring due to the metabolic imbalances of gestation.

The present study was an attempt, using the rat as a model,
to determine whether male offspring subjected to the
metabolic alterations of a diabetic pregnancy would show
altered sexual behavior in adulthood.

Long Evans females (60-70 days) were adapted to the
lab and then given a single intravenous injection of either
a diabetogenic agent, streptozotocin, or a control vehicle,
buffered citrate. Females were then mated and weight

and urine sugar were measured on days 1, 7, l4, and 20 of

Patricia Hilley Ruppert

pregnancy. Pups were weighed on the day of birth and
litters were culled to 8 pups; some litters were cross
fostered and others were reared by their natural mothers.
Weight and urine sugar were measured in the male offspring
at 50 and 90 days.

Beginning at 60 days, each male was given three
weekly pretests for sexual behavior with a receptive female;
this established a baseline of sexual activity. Then 3
weekly sexual behavior tests, also 30 minutes each, were
given and frequencies and latencies for mounts, intro-
missions and ejaculations were recorded. Ten days to 3
weeks after the last test, each male received a satiation
test, with a criterion of 30 minutes without an intro-
mission for termination of the test.

Male offspring of diabetic mothers, either reared
by their own mothers or cross fostered to control mothers,
showed a shortened latency to the initiation of sexual
behavior. A change in temporal pacing resulted in
significantly shorter latencies to intromission and
ejaculation. The decreased intromission latency shown by
these males when compared to controls indicates a lowering
of the copulatory threshold for the initiation of sexual
behavior. The decrease in ejaculation latency and decrease
in intromission frequency in offspring of diabetic mothers
indicates a concomitant lowering of the ejaculatory

threshold.

Patricia Hilley Ruppert

The pattern of sexual behavior shown by male off-
spring of diabetic mothers is also seen in adult male rats
subjected to various manipulations, all of which increase
arousal level: castration, adrenalectomy, audiogenic
stimulation, handling of rats during copulation, peripheral
electrical shock and rewarding or aversive brain stimulation.
Changes in patterning in sexual behavior are maladaptive
in decreasing reproductive efficiency. A species specific
pattern of number and spacing of intromissions is needed
to initiate neuroendocrine reflexes for the initiation of
pregnancy. It is possible that behavior shown by offspring

of diabetic mothers would be detrimental in this regard.

SEXUAL BEHAVIOR IN MALE OFFSPRING

0F FEMALE DIABETIC RATS

BY

Patricia Hilley Ruppert

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF ARTS

Department of Psychology

1976

ACKNOWLEDGMENTS

I would like to express appreciation to my
committee members Dr. Lynwood G. Clemens, Dr. Lawrence I.
O'Kelly, and Dr. Glenn I. Hatton for their individual and
thoughtful contributions to the shaping of this thesis.

The author was supported by a NIMH Traineeship,

5 T01 MHlOGll, and the research was supported by NIH,

HD 06760-09 to Dr. Lynwood G. Clemens.

ii

TABLE OF CONTENTS

Page
LIST OF TABLES O O O O O O O O O O O O 0 iv
INTRODUCTION. . . . . . . . . . . . . . 1
Diabetes Mellitus . . . . . . . . . . . 2
Impotence in Males . . . . . . . . 6
Sex Differences in the Diabetic Rat . . . . . 8
Streptozotocin . . . . . . . . . . . . 13
Diabetic Pregnancy . . . . . . . . . . . 15
Masculine Sexual Differentiation and BehaVior
in the Rat O O O O O O O O O O O O O 1 8
Statement of the Problem . . . . . . . . . 22
MTHOD. O O O O O O O 0 O O O O O O O 2 5
Anin1als O O O O O O O O O O O O O O O 25
Treatment . . . . . . . . . . . . . . 25
Testing 0 O O O O O O O O O O O O I O 27
RESULTS 0 O O O O O O O O O O O O O O 29
Pregnancy . . . . . . . . . . . . . . 29
Offspring . . . . . . . . . . . . . . 30
Male Behavior Tests. . . . . . . . . . . 31
D1 SCUSSION O O O O O O O O O O O O O O 3 8
sexual Arousal O O O O C O O O O O O O 39
Adaptive Significance . . . . . . . . . . 42
EtiOlogy O O O O O O O O O O O I O O 4 3
Conclusion. . . . . . . . . . . . . . 44
APPENDICES
Appendix A O O O O O 0 O O O O O O O O 46
Appendix B . . . . . . . . . . . . . . 47
REFERENCES 0 O O O O O O O O O O O O O 49

iii

Table

1.

4.
5.
6.

LIST OF TABLES

Mean Litter Size, Mean Weight Per Litter, and
Mean Percent Males Per Litter From Diabetic
and Control Mothers . . . . . . . .

Mean Weight of Male Offspring at 90 Days. .

Proportion of Copulators During Weekly Mating
Tests and Satiation Test . . . . . .

Mean Behavioral Scores on Weekly Mating Tests

Mean Behavioral Scores on Satiation Test. .

Levels of Significance on Mann-Whitney U Test
for Behavioral Measures on Weekly Mating

Tests and Satiation Test . . . . . .

Urine Sugar and Body Weight for Mothers . .

Urine Sugar and Body weight for Male Offspring.

iv

Page

30
31

32
33
35

37
46
47

INTRODUCT ION

Complications of diabetes mellitus encompass a wide
spectrum of pathology, ranging from the obvious disruption
of carbohydrate metabolism to retinopathy and neurological
lesions. One concomitant of diabetes mellitus frequently
seen in the clinic is impotence in adult males. While the
average incidence of this disorder is 50 percent in diabetic
males, its etiology remains undetermined. In male rats
made diabetic in the laboratory there are disturbances in
testicular functioning similar to those seen in the human,
and fertility is reduced. However, there have been no
published studies on the patterning of sexual behavior in
these males. In a preliminary investigation in Dr. Clemens'
laboratory, Mary Hoover (unpublished) studied the sexual
behavior of male rats made diabetic with streptozotocin
(STZ) injections. She found an increased number of mounts
(poorly oriented), and a decreased frequency of intro-
missions and ejaculations. Latency to erection was also
increased.

Offspring of a diabetic mother might also be

expected to have impaired sexual functioning in adulthood.

Although the offspring themselves are not overtly diabetic,
the metabolic stress of hyperglycemia and hyperinsulinemia

in utero might impair the develOpment of normal male sexual
potential. 'In this thesis, I aim to study this problem by
making female rats diabetic with STZ injections, impregnating
them and testing the sexual behavior of their male off-
spring in adulthood.

In the remainder of this section, I shall elaborate
on the rationale for this experiment. The sections are as
follows: first a brief description of naturally occurring
diabetes mellitus, and the disturbances of sexual functioning
in the human male. Following is a summary of the incidence
of experimental diabetes in male and female rats, and the
effect on male reproductive capacity. Then I will describe
the diabetogenic action of streptozotocin, the dynamics of
diabetic pregnancy in the rat, and the development and
expression of the normal patterning of sexual behavior in

the male rat.

Diabetes Mellitus

 

Diabetes is a metabolic disorder currently affecting
five million Americans, and is the fifth leading cause of
death by disease in the United States today. The highest
incidence rate is for the obese or those with a family
history of diabetes. The disease can be roughly divided
into two divisions: growth onset, occurring before puberty,

and maturity onset. The growth onset type is usually more

severe: insulin must be used for maintainence rather than
the oral hypoglycemic agents and these patients are more
prone to ketosis and other complications of diabetes.

The course of the disease in an individual usually
progresses through four stages: prediabetes, latent
chemical diabetes, chemical diabetes, and overt diabetes.
Diabetes is based on abnormal blood glucose concentrations
either in the fasting state or after a glucose stimulation
test. Onset in younger patients is usually rapid and is
accompanied by polyuria, thirst, weight loss, increased
appetite and visual disturbances. If these early symptoms
are not recognized and treated, acidosis and coma may
result. In gradual onset diabetes, the symptoms are fewer
and complaints of nocturia, muscle cramps or impotence may
lead to diagnosis of diabetes (Gorman, 1973).

The mechanisms responsible for diabetes mellitus
are still not fully understood; models of insulin action in
the normal individual and the hormonal interrelationships
contributing to hyperglycemia are currently being revised
and reformulated. Insulin seems to achieve its anabolic
actions by binding to the cell membrane and changing its
structural conformation, resulting in release of calcium
ions from high affinity binding sites. The increased
concentration of intracellular calcium then functions as a
second messenger to alter the sensitivity of protein kinase
to cAMP, eventually resulting in activation of glycogen

synthetase for conversion of glucose to glycogen. The same

mechanism would result in activation of pyruvate dehydro-
genase to stimulate lipogenesis (Kisselbah, Tulloch, HOpe-
Gill, Clarke, Vydelingum & Fraser, 1975; Larner, 1975;
Lazarus & Davis, 1975). In diabetes mellitus, an insuffi-
ciency of insulin results in high levels of glucose in the
blood and urine, breakdown of protein with urinary excretion
of nitrogen, and breakdown of lipids and fatty acids
resulting in accumulation of ketone bodies.

Complications of diabetes mellitus can result from
vascular or metabolic abnormalities. Acute metabolic
complications are reversible by a readjustment of the
insulin supply to the body. Currently two approaches are
being investigated to improve long term control of blood
sugar. The first involves transplantation of donor or
artificial pancreases, or transplantation of islets of
Langerhans. These attempts have not been successful to date.
The second approach aims at modulating the hormonal
imbalances in the system.

Insulin deficiency alone is not responsible for the
hyperglycemia of diabetes. The current hypothesis is that
the metabolic abnormalities result from an absolute or
relative hypoinsulinemia and an absolute or relative
hyperglucagonemia. A deficiency of insulin is responsible
for underutilization of available glucose and the excess
production of free fatty acids. The increased glucagon
secretion is responsible for hepatic overproduction of

glucose (Unger & Orci, 1975). This was discovered through

the use of somatostatin, a polypeptide isolated from the
hypothalamus which inhibits the release of growth hormone
from the pituitary, and also lowers plasma insulin and
glucagon levels when infused. During the period of infusion,
plasma glucose is also lowered. Somatostatin in itself

has no effect on muscle glucose uptake or hepatic glucose
production; reduction in plasma glucose was attributed then
to the inhibition of glucagon stimulation of hepatic glucose
production (Alberti, Christensen, Christensen, Hansen,
Iversen, Lundbaek, Beyer-Hansen, & Orskov, 1973; Koerker,
Ruch, Chideckel, Palmer, Goodner, Ensencke, & Gale, 1974;
Gerich, Lorenzi, Hane, Gustafson, Guillemin & Forsham, 1975:
Sakurai, Dobbs & Unger, 1975). Analogues of somatostatin
are being synthesized for clinical use in the hope that a
better regulation of blood glucose can minimize the compli-
cations of diabetes.

Diabetes still presents special problems for the
pregnant woman. Although fertility is normal in diabetic
females, fetal mortality is high. Complications are usually
related to the severity of the diabetes, and the probability

of fetal viability is classified as follows:

Class A: Mild chemical diabetes (fetal survival
approaching normal)

Class B: Overt diabetes of less than 10 years which
developed in adult 1ife--no complications
(67% fetal survival)

Class C: Diabetes beginning between 10 and 19 years or
diabetes of 10 to 19 years duration--no
vascular disease (48% fetal survival)

Class D: Diabetes of more than 20 years with a high
probability of vascular disease; diabetes
beginning before 10 years or diabetes associ-
ated with mild retinopathy or leg-vessel
calcification (32% fetal survival)

Class E: Pelvic vessel calcification on x—ray (13%
fetal survival)

Class F: Diabetic nephropathy (3% fetal survival)
Class R: Severe diabetic retinopathy (high fetal loss)
Effects on the fetus include a slightly increased
risk of malformations, hypertrophy of the islets of
Langerhans under the stimulation of maternal hyperglycemia,
and a large body size resulting from excessive accumulation

of fat and fluid, but also larger organ size (Gorman, 1973).

Impotence in Males

 

The average incidence of this disorder is estimated
to be 50% of diabetic males (Ellenberg, 1971; Faerman,
Vilar, Rivarola, Rosner, Jadzinsky, Fox, Perez-Lloret,
Bernstein-Hahn & Saraceni, 1972; Kolodny, Kahn, Goldstein &
Barnett, 1974). When this disorder does develop, the
symptomology follows a characteristic pattern. Impotence
usually occurs within five years of the occurrence of
diabetes (Rubin & Babbott, 1958; Schoffling, Federlin,
Ditschuneit & Pfeiffer, 1963; Kolodny §E_al., 1974); in
some cases the impotence may precede the diagnosis of
diabetes. The onset of impotence is usually gradual with
decreased firmness of erection within six months to one
year; 53% of the impotent males in one study had no morning

erections and were unable to masturbate although libido

persisted, indicating that the impotence is not of psycho-
genic origin (Rubin 22 31., 1958). Nocturnal emissions
are usually absent also. No correlations have been found
with the age at onset, nor in some studies with the compli-
cations of the disease, either vascular or neurological.

The etiology of impotence remains unknown. Schoffling
25 El! (1963) has been a main influence attributing impotence
and sterility to secondary hypogonadotropic hypogonadism.
They found reduction in testicular size and consistency,
decreased prostate size and volume of seminal fluid,
abnormal sperm counts, decreased seminal fluid fructose,
decreased gonadotropins and atrophy of the seminiferous
tubules with basement membrane thickening. With combination
therapy with chorionic gonadotropin and testosterone, the
impotence disappeared and semen analysis, sperm counts and
prostate size improved. This would indicate that the
impotence was a result of hormonal deficiency (Schoffling,
1965). However, later studies have found no difference in
plasma testosterone concentrations between potent and
impotent diabetics and normals (Ellenberg, 1971; Faerman,
et 31., 1972; Kolodny, gt $1., 1974) although slight to
marked hypospermatogenesis was observed. Testosterone
therapy alone or in combination with chorionic gonadotrOpin
in a range of doses and treatment regimes failed to
ameliorate the condition. A possible explanation of the

discrepancy is that Schoffling used a very narrow range of

normal values as indices of function but did not test for
plasma testosterone.

A link has been made, however, between impotence
and neuropathy in diabetes (Ellenberg, 1971; Faerman,
23 31., 1972; Kolodny, 23 31., 1974). In a survey of
diabetic males, Ellenberg found that 59% were impotent and
of these, 88% had some accompanying neuropathy. Of the 41%
diabetics with normal potency, only 12% had some neuro-
pathological disorder. This pathology consists of diminished
nerve conduction velocity, abnormal deep tendon reflexes
and especially bladder dysfunction. Since autonomic path-
ways for micturition and erection are identical, involving
the second, third, and fourth components of the spinal
cord, a neurological lesion might be responsible for
impairment of both functions. This has not yet been
demonstrated conclusively. Recently, Faerman, Glocer, Fox,
Jadzinsky and Rapaport (1974) studied the nerve fibers of
the corpora cavernosa in five impotent and five non-impotent
diabetics. Four of the five impotent and none of the
control diabetics showed irregularities consisting of
diffuse thickenings of the nerve fiber. This would indicate
that the reflex arc of erection was damaged at a peripheral

level.

Sex Differences in the Diabetic Rat'

 

Diabetes has been studied in the rat as a phenomenon

induced by sub-total (95%) pancreatectomy, alloxan, STZ and

other preparations. Disturbed sexual functioning, i.e.,
reduced fertility has been noted in noted in male rats
following these treatments. Foglia, Schuster and Rodriguez
(1947) first described a sex difference in the occurrence
of diabetes after sub—total pancreatectomy in the rat.
Following a seven hour fast, 92% of the males and only 28%
of the females had blood sugar levels over 150 mg%. Females
after surgery showed a slow linear increase in the number
of diabetics up to six months, while the males showed an
abrupt increase between one and two months, continuing at

a high number to six months. Paired feeding of males and
females showed that the higher incidence of diabetes in
males was not due to a higher food intake which might
aggravate the condition.

Castration of males simultaneously with pancreatectomy
caused a slight reduction in the incidence of diabetes,
however, castration after the onset of diabetes was in-
effective. Testosterone in doses of 1—5 mg/day for 10
days had no effect in either the prediabetic or diabetic
phase (Foglia, 33 31., 1947). Angerwell, Hesselsjo,

Nilsson and Tisell (1967) treated alloxan diabetic castrates
and non-diabetic castrates with testosterone propionate
(TP), .5 mg/day for two weeks. Animals were castrated at a
weight of 62 grams and alloxanized 30 days later. Prior
to TP treatment, both groups of castrates showed inhibition
of the ventral prostate, dorsolateral prostate, coagulating

glands and seminal vesicles; there was no difference in

10

this reduction between alloxan diabetic and non-diabetic
castrates, indicating that insulin deficiency was not a
factor. While TP treatment markedly stimulated the growth
of these organs in both groups, the increase was of lower
magnitude in the diabetics, indicating slightly less
secretory capacity. This could be a direct result of
insulin deficiency or a reduced output of other synergistic
hormones, such as growth hormone or prolactin.

Female rats who were pancreatectomized and ovari-
ectomized eight to 10 days later showed a higher incidence
of diabetes than after pancreatectomy alone (Foglia, 33 31.,
1947). Androgenized females who were given TP, 1 mg/rat
on day 3, and then pancreatectomized on day 60 had a higher
fasting blood sugar level at 30 and 60 days post-
pancreatectomy than controls (Foglia, Basabe, & Chieri,
1969). Neonatal castration of males and females decreased
the incidence in males and increased the incidence in
females of overt diabetes; this effect was more pronounced
than in adult castrations, with the incidence intermediate
between that of normal males and females (Foglia, Borghelli,
Chieri, Fernandez-Collazo, Spindler, & Wesely, 1963).
Estradiol benzoate (EB) given in doses of 2, 4, or 15 ug/day
for six months exerted a protective effect, i.e., normal
beta cell granulation, on both castrated and intact pan-
createctomized males and females (Foglia, 33 31., 1947).
Using a criterion of three months normal blood sugar without

the use of insulin after the cessation of EB treatment,

ll

Rodriguez (1954) found that with prior treatment with EB
alone, 47% were normalized and in conjunction with insulin
69% were normalized. This effect was achieved in females
whose blood sugar was less than 250 mg%.

The above results seem to indicate that the ovary
rather than the testis is instrumental in the normalization
of diabetes; removal of the testes in the adult male only
slightly decreases the incidence of diabetes while removal
of the ovary in the adult female greatly increases the
incidence. EB exerts a protective influence not only on
the female but also on the male. Whether this estrogenic
influence represents an effect on pituitary secretions, the
adrenal cortex, metabolic action on the liver or a direct
effect on the islet cells themselves is not known. Electron
micrographs have established that besides mature secretory
granules in the pancreas (260 nm) which are affected by STZ,
there remain some immature granules which persist (65 nm)
(Arison, Ciaccio, Glitzer, Cassaro, & Pruss, 1967). These
could represent sites of estrogen facilitation of insulin
release. Goodman and Hazelwood (1974) injected alloxan
diabetic rats for 10 days with 10 ug EB per day to assess
its effect on diabetes. In the minimally diabetic,
urinary glucose excretion was decreased although plasma
glucose was not changed. They also found increased plasma
growth hormone and corticosteroid levels with slightly
enlarged pancreatic islets. It seems that given a sufficient

number of beta cells, estrogen can stimulate these cells

12

directly, although a modification of hypophyseal release
of growth hormone and adrenocorticotropin could also be
involved.

What are the implications of these findings for the
sexual behavior of the diabetic rat? Foglia 33 31. (1963)
found that in the period of manifest diabetes, the ability
of males to impregnate females drastically decreased
depending on the severity and duration of the diabetes.
Males with blood sugar levels of 150 to 200 mg/ml impregnated
45% of the females they were mated with; this compares with
50% for the laparotomized controls. Rats with higher fasting
blood glucose levels only impregnated 12% of the females.
Similarly, with a zero to two week duration of diabetes,

49% of the females were impregnated by diabetic males but by
41 to 44 weeks of diabetes none of the males were able to
impregnate females. In conjunction with this sterility,
diabetics with blood glucose levels over 300 mg/ml showed
reduced body weight, testis, prostate, seminal vesicles

and coagulating gland weight. This was accompanied by
tubular alterations, with a delay in spermatogenesis.
Fecundity was reduced to a greater extent if these lesions
were present.

In immature rats (40-44) days, alloxan diabetes
reduced testis diameter and tubule diameter, prostate and
seminal vesicles secretion and delayed descent of the testes
depending on the severity of the diabetic state. Permanent

diabetes resulted in sterility but treatment with insulin,

13

TP or chorionic gonadotropin, alone or in combination were
able to normalize prostate and seminal vesicle secretions,
and to a lesser extent, the testicular and tubule diameters
(Hunt & Bailey, 1961). Schoffling, Federlin, Schmitt and
Pfeiffer (1967) found no change in spermatogenesis with mild
alloxan diabetes with blood sugar from 151 to 250 mg/ml.
Moderate diabetes interfered with spermatogenesis and severe
(351-450 mg/ml) caused absence of Leydig cells, decrease in
the volume of germinal epithelium and absence of spermatazoa
and spermatids. There is no evidence to date on the pattern
of sexual behavior in these rats which would be correlated

with disturbed endocrine functioning and sterility.

Streptozotocin

 

Streptozotocin, an antibiotic isolated from
Streptomyces achomogenes, was reported by Rakieten,
Rakieten and Nadkarni (1963) to have diabetogenic properties
along with its anti-tumor activity. Since then a number of
studies have shown the uniformity and specificity of STZ
action. It is superior to alloxan in inducing experimental
diabetes in having greater selective toxicity for beta
cells, a larger dose range and an insensitivity to nutri-
tional state in its action (Junod, Lambert, Stauffacher &
Renold, 1969).

The major action of STZ, as studied by light and
electron microsc0py, is a rapid and irreversible degranula-

tion of the beta cells of the islets of Langerhans (Rakieten

14

33 31., 1963; Arison 33 31., 1967; Junod, Lambert, Orci,
Pictet, Gonet & Renold, 1967). Rakieten and Junod attri-
buted this to necrosis of the beta cell seen within seven
hours whereas Arison found degranulation only indicating
that insulin secretion or production was inhibited. Whether
actual necrosis accompanies degranulation is still an open
question.

The effect of STZ is achieved within 48 hours of
intravenous administration. The minimum effect dose is
35 mg/kg and LD50 is 137.7 mg/kg, with increased glycosuria
and ketonuria with increased dosage (Rakieten 31 31.,
1963; Junod 33 31., 1969). The initial effect of moderate
doses of STZ, 50 to 65 mg/kg, is a marked increase in blood
glucose two hours post-injection along with a rise in free
fatty acids and blood ketone bodies. This is followed by
a marked hypoglycemia at seven to 10 hours, with a decrease
in blood ketone bodies and free fatty acids. This hypo-
glycemic response is directly related to an increase of
insulin released from the pancreas as a result of beta cell
degranulation. Between 24 and 48 hours, a permanent
hyperglycemia is established with a return to normal
fasting blood ketone levels; STZ does not produce ketonuria
at low doses. Pancreatic insulin levels are decreased to
5% of normal after 24 hours and after one month remain at
less than 2% of normal. Glycosuria begins on the first
day and increases throughout the first week. This is

accompanied by polydipsia and polyuria, with urine volume

15

increasing from control levels of 1-5 ml/24 hours to
100-150 m1/24 hours (Junod 33 31., 1967; Schein, Alberti &

Williamson, 1971).

Diabetic Pregnancy

 

Pregnancy involves a state of metabolic stress for
the normal female, and this condition is exacerbated in the
diabetic female. Normal pregnancy in the rat is character-
ized by a fall in blood sugar starting between days 10 and
15 of pregnancy, with an increase in the insulin content of
the pancreas and a marked increment in the response of beta
cells to induced glucose stimulation (Malaisse, Malaisse-
Lagae, Picard & Flament-Durand, 1969; Rishi, Golob, Becker &
Shah, 1969). The total pancreatic insulin content in late
pregnancy is 53% greater than that of non—pregnant controls.
Malaisse 33 31. found a smaller increase of 15%. These
results indicate increased secretory capacity during late
pregnancy.

Although insulin does not cross the placental
barrier, during this time the fetal pancreas is developing
its own secretory capacity. On the twelfth day of gestation
the islet tissue of the fetal pancreas is undifferentiated
but by day 21 maximum growth and differentiation has been
achieved. By day 18, the duct cells represent 40% of the
pancreatic volume and less than 1% is islet tissue. On
day 22, the proportions of 2% islet tissue and 1% duct
tissue resemble those of the adult (Rishi, 33 31., 1969;

Hegre, McEvoy, Bachelder & Lazarow, 1973).

16

This secretory pattern is drastically altered both
in the mother and in the fetus by the administration of
diabetogenic agents. Common results of such treatments are
sterility, abortion, reduced litter size, fetal abnormalities
and either an increase or decrease in fetal weight. Most
studies report higher fetal mortality (Kim, Runge, Wells &
Lazarow, 1960; Golob, Rishi, Becker & Moore, 1970; Lazarow &
Heggestad, 1970; Pitkin, Plank & Filer, 1971; Sybulski &
Maugham, 1971). This is not due solely to hyperglycemia
since it was also found in studies of sub-diabetic rats
(Lazarow, Kim & Wells, 1960). At least one study has shown
a predominance of female offspring with 46% in normal litters
and 64% in litters of pancreatectomized mothers (Foglia,
1970). This effect is also seen in sub-diabetic female
rats but is not observed in offspring of diabetic fathers,
indicating that it is environmentally induced.

Some studies report fetuses with decreased birth
weight (Golob, 33 31., 1970; Sybulski, 33 31., 1971) while
others report cases of fetal giantism (Lazarow, 33 31,,
1960; Foglia, 1970; Pitkin, 33 31., 1971). Lazarow and
Heggestad (1970) suggest this discrepancy is due to differ-
ences in gestational age; when litters are delivered at
519 hours (the earliest estimated time for Spontaneous
delivery) they tend to be lighter, but when spontaneous
delivery occurs, usually after a prolonged gestational
period, the fetuses are heavier. Pitkin and VanOrden

(1974) attribute the differences in weight, still a

17

controversy, to the severity of the diabetes, with higher
glycosuria associated with smaller fetuses.

What effect does maternal hyperglycemia.have on
the fetal pancreas? This effect follows a pattern of
maternal hyperglycemia and fetal insulinemia as described
by Pedersen (1967). The fetal beta cells are subjected to
high blood sugar levels in utero, resulting in degranulation
of the beta cells and insulin depletion with glycogen
infiltration. When pancreatic segments from normal rat
embryos are grown in vitro, the number of beta granules is
inversely proportional to the level of glucose in the
media. When fetal pancreases from diabetic mothers are
explanted at day 19.5 or 21.5, they contain only 5%
granulated beta cells versus 75% for controls. On a low
glucose media, however, rapid granulation can be observed
in two days (Wells, Schweisthal, Marx, McKay, Saccoman &
Lazarow, 1967; Wells & Lazarow, 1967). In vivo, blood sugar
returns to normal levels in the neonate within 24 hours
postpartum, although the appearance of the beta cells is not
completely normalized for three to 10 days. Although none
of the offspring studied developed overt signs of diabetes,
they exhibited abnormal glucose tolerance curves for the

two years they were studied (Lazarow, 33 31., 1970).

18

Masculine Sexual Differentiation
and Behavior in the Rat

 

 

The development of normal sexual capacity in the
male rat is contingent upon the presence of androqen during
the critical perinatal period of sexual differentiation.
Fetuses are bisexual at 17 days of gestation; the anlagen
of both male and female reproductive structures are present.
Under the influence of the sex chromosomes in the male,
through an as yet unexplained inductive substance, cortexin,
the cortex of the undifferentiated gonad develops into a
testis and the medulla regresses. In the presence of the
fetal testes, the Wolffian ducts develop the male accessory
sex organs, and the Muellerian ducts are inhibited. In the
final step of morphological development, the genital
tubercle develops as the penis (Jost, 1972). During this
period, the potential for developing masculine behavior
patterns is established.

The plasticity of the behavioral system allows for
a period of up to 10 days postnatally for behavioral
differentiation to occur. In the presence of androgen
during this period, both genetic males and females will
show a high probability of exhibiting a male pattern of
sexual behavior. Conversely, in the absence of androgens
or other gonadal steroids, both genetic males and females
will show a high probability of showing lordosis, the
female pattern of receptivity to the male. Beyond this

period of maximal sensitivity to the action of steroids,

19

removal of endogenous androgen does not inhibit masculine
behavior potential, given a sufficient supply of androgen

in adulthood. However, androgen deprivation perinatally
results in a deficiency of male sexual functioning in
adulthood regardless of the level of circulating androgen in
the adult system.

According to this formulation, then, two periods of
androgen action are critical: perinatally to organize
behavioral patterns and in adulthood to exhibit the behavior
(Young, Goy & Phoenix, 1964). In this theory of andro-
genization of behavior, the hormones are presumed to act in
an organizational way on undifferentiated neural tissues as
they do on the genital tract. This would result in the
develOpment of an acyclic pattern of gonadotroPin release
characteristic of the male. In the adult, gonadal hormones
would act via excitation or inhibition to regulate the
gonadotropin secretion and thus, the hormone dependent
patterns of sexual behavior (Harris, 1964).

Experimental evidence supports the necessity of
early androgen. When male rats are castrated on day 1, they
are incapable of showing the ejaculatory response in adult-
hood. When castration occurs on day 5, from 8.3% to 25% of
the males show an ejaculatory response while 100% of intact
males ejaculate (Grady, Phoenix & Young, 1965; Gerall,
Henricks, Johnson & Bounds, 1967). Nadler (1969) has also
administered an anti-androgen, cyproterone acetate, to

pregnant female rats and then castrated their offspring on

20

day l to eliminate both prenatal and postnatal effects of
androgen. When tested with testosterone prOpionate in
adulthood, none of these males ejaculated. In both types
of studies, males exhibited high levels of lordosis behavior
when treated with appropriate hormones; this provides
corroboration that lack of androgen exposure during
development leads to a high probability of female behavior
patterns. However, it is significant to note that males
depriVed of early androgen also have reduced penis length
which could also alter the sensory feedback derived from
copulatory behavior. Beach, Noble and Orndoff (1969) have
shown that unless androgen is present before day 10, the
penis will not grow in response to later androgen treatment.
Therefore, they offer an alternative hypothesis to the
organizational action of androgen action on neural structures.
Mechanisms for masculine behavior patterns may be determined
by prenatal androgen but in the absence of normal penile
development this behavior cannot be normally expressed.
The crucial experiments have not been done to determine
peripheral and central factors.

Expression of sexual behavior in adulthood follows
a stereotypic temporal patterning. Initial encounters
between a male and female usually involve exploratory
anogenital sniffing as a component of precopulatory
behavior. This is followed by a mount, i.e., the male
Clasps and palpates the sides of the female with his fore—

limbs while thrusting the pelvic region. These movements

21

can be aborted by unreceptive female, and in any event, the
male slides weakly off her back. With intromission,
palpation and thrusting culminate in a deep thrust penetra-
ting the vagina which terminates in the male lunging back-
wards and ending contact with the female. An ejaculation
can be differentiated from an intromission in that the
final lunge is not executed; the male remains clasped to
the female and then slowly raises his forelegs and drOps
off. Preceding each segment of activity, the male chases
the female who is actively h0pping and darting as a means
of solicitation, and she then remains immobile for the
duration of the mount, intromission or ejaculation (Young,
1961).

Each intromission lasts approximately .5 seconds
with an interval of 30 to 60 seconds between intromissions.
The first ejaculation occurs after approximately eight to
15 intromissions while four to seven intromissions are
required for subsequent ejaculations in a series. The post-
ejaculatory interval, the period following an ejaculation
during which the male is unresponsive to further sexual
stimulation, lasts approximately five minutes after the
first ejaculation. Studies of sexual exhaustion or
satiation have shown that the post-ejaculatory interval
increases with subsequent ejaculatory series. The
ejaculation latency, intromission frequency and inter-
intromission interval decline after the first ejaculation

and then rise as satiation is approached. A recovery

22

period of about two weeks is required for the male to
return to the initial temporal patterning of the first
ejaculation (Beach & Jordan, 1956; Larsson, 1956; Bermant,

1967; Dewsbury, 1968).

Statement of the Problem

 

In this introduction, I have elaborated on the
factors which might influence the sexual behavior of male
rats born to diabetic mothers. Evidence was presented
indicating a correlation between the occurrence of diabetes
and sterility in the adult male. This could be the result
of a hormonal deficiency in either of two ways. A direct
effect could be a decrease in the levels of circulating
testosterone or an insensitivity of the target tissues to
the androgen. Secondarily, androgen sensitive reflex
systems might be compromised due to vascular and neuro-
logical complications of the diabetic state.

The perinatal period has also been demonstrated to
be sensitive to androgen deficiency. Males exposed to the
gestational period of a diabetic pregnancy are vulnerable
in two ways. First, there might be a direct decrease in
the testosterone production of the fetal testis as a result
of exposure to the diabetogenic condition of the mother.
This would parallel the exposure of the adult male in
which the expression rather than organization of sexual
behavior is inhibited. Also, differentiation of sexual

behavior might be impaired by the diffuse effects of

23

continued metabolic stress of hyperglycemia and hyper-
insulinemia experienced throughout the pregnancy by both
the mother and her pups.

The problem I wish to consider in this study is
whether the process of sexual differentiation in male rats
will be disrupted by the hyperglycemia and hyperinsulinemia
experienced by the offspring of a diabetic mother. It has
been shown that organization of male sexual behavior is
impaired by imbalances of gonadal steroids, and also that
expression of sexual behavior is disrupted by diabetes.
This study will determine if the same processes which can
affect sexual behavior in adulthood can also affect the
organization of sexual behavior neonatally.

The following hypotheses are offered concerning the
sexual behavior of the male offspring:

1. Adult behavior may be disrupted in offspring of
diabetic mothers either directly through an insufficient
supply of androgen during the period of sexual differ-
entiation or indirectly through the deleterious effects of
prenatal metabolic stress. Disruption of sexual behavior
would be characterized by a high number of mounts
(reflecting an inability to achieve vaginal penetration), a
decreased inter-intromission interval (reflecting the
decreased excitatory value or sexual stimulation) and a
decreased number of ejaculations to satiation (reflecting

decreased sexual motivation).

24

2. An alternative hypothesis is that male offspring
of diabetic mothers will exhibit normal sexual functioning
in adulthood despite the aberrant intrauterine environment.
Previous studies on the effects of stress during pregnancy
have been confined to the study of emotional responses with
the exception of one study on the effect of exogenous
stress on sexual behavior. Since there are no systematic
data available on the effects of stress on sexual differ-
entiation it is probable that fetuses are able to come
pensate in varying degrees to different categories and
intensities of trauma. Prolonged hyperglycemia may create
an altered homeostatic condition for the fetus. Pups might
also differentiate normally given decreased testosterone
levels if the amount produced is still in excess of the

sensitivity of the target tissues.

METHOD

Animals

Long Evans (Charles River) virgin female rats, aged
70 to 90 days, were used as mothers in this experiment
after an adaptation period to the laboratory of approxi-
mately one week. They were housed individually in cages
measuring 36x30.5xl7 cm. Purina Lab Chow and water were
available ad lib, and a constant temperature was maintained.
All animals were under a 14-10 hour reversed light-dark
cycle, with lights off at 1100 hours. Pups were weaned at
21 days, and the males were housed by litter in cages
measuring 15x35.5x22.5 cm. During urine collection,
animals were housed in individual metabolic cages under

the same conditions.

Treatment

 

All females were placed in metabolic cages for a
24 hour urine pretest. Urine samples were tested for
glucose using two indices. Clinitest (Ames Company) which
indicates the presence of reducing substances, resonds to
minimum levels of 150 mg/ml. Testape (Eli Lilly Company),
a specific test for glucose, can discriminate between 0,

100, 250, 500, and 2000 mg/ml. Acetest (Ames Company)

25

26

was used to test for ketonuria, an index of the severity of
diabetes. A minimum positive reaction indicates the
presence of at least 10-20 mg/ml acetoacetic acid. A
moderate reaction indicates twice that concentration, and
a strong reaction indicates over 50 mg/ml acetoacetic acid
(Pyke, 1968).

Following this procedure, females were anesthetized,
ear tagged, weighed, and given an intravenous injection of
40 mg/kg STZ. The STZ (National Cancer Institute) was
administered in a 4% solution in 0.4M citric acid with the
pH adjusted to 4.0 with sodium hydroxide. Controls received
an equivalent volume of buffered citrate solution. All
solutions were prepared at the time of injection. After a
two day recovery period, females were placed daily with a
male until mating took place. This was considered day l
of pregnancy. Only females who were severely diabetic as
judged by maximum readings throughout pregnancy on the two
indices of urine sugar were included in the study.

On days 1, 7, l4, and 20 of pregnancy, glycosuria
and acetonuria were measured from urine samples. Weights
also were recorded. Starting on day 21, all cages were
checked daily for the presence of litters. The date of
birth, and the number, sex and weight of the offspring were
recorded. All litters were culled to eight pups, a mixture
of both males and females, between the first and third day.
Some litters were cross fostered to assess possible effects

of postnatal maternal influences.

27

Testing

Males were weighed and tested for glycosuria and
acetonuria at 50 and 90 days, and following the final
behavioral test. Litters of experimental and control
animals were numbered randomly so the behavioral tests were
conducted without experimenter knowledge of the treatment
group of the animal. Testing for male sexual behavior began
at 90 days of age. Testing was conducted in clear plexi-
glass arenas (59x46.5x51.5 cm) in a dimly lighted room
between 1300 and 1700 hours. Males were placed in the
arena for a five minute adaptation period before testing.

At the start of the test period a receptive female (screened
with a normal male) was placed in the test arena. Recepti-
vity was induced in the females with 12 ug estradiol
benzoate for three days followed by .5 mg progesterone on
the fourth day, four hours prior to testing. Mounts (with
pelvic thrusting), intromissions (with vaginal penetration)
and ejaculations were recorded on an Esterline Angus Event
Recorder.

Each weekly test was conducted for 30 minutes.

Three weekly pre-tests were followed by three tests in
which the following behaviors were scored: mount latency,
intromission latency, ejaculation latency, mount frequency,
intromission frequency, inter-intromission interval, and
post-ejaculatory interval for the first series. Total
number of ejaculations per test also was recorded. Ten days

after the last weekly test, each animal was tested for

28

satiation. This was defined as one half hour after the
test had begun without an intromission. The total number
,of ejaculations preceding satiation was a measure of the
upper limit of the animal's performance.

All data were analyzed non-parametrically by either

a Kruskal-Wallace Test or a Mann-Whitney U Test.

RESULTS

Pregnancy

 

Present findings confirm those reported in the
literature in that diabetic females had a slightly pro-
longed gestational period. Also, while all of the control
mothers reared their litters to weaning, pups from four
diabetic mothers had to be sacrificed prior to weaning
because of their emaciated condition. These diabetic
mothers showed maternal behavior by aggregating the young
in a nest and crouching over them, but it is likely that
the diabetic condition interfered with milk letdown.

Appendix A contains the weights, urine sugar and
ketosis measurements for both severely diabetic and control
mothers on days 1, 7, l4, and 20 of pregnancy. Severely
diabetic mothers showed urine sugar measurements of 1 to 2%
throughout pregnancy. This was the maximum reading on both
of the semi-quantitative indices of urine sugar, Clinitest
and Testape. Control mothers showed minimal levels on
these indices; this could be attributed to normal fluctuation
or to some unavoidable contamination of the urine with the
food pellets. No animals showed a positive reaction with

Acetest at any time. The mean weight gain during pregnancy

29

30

was 159.7 gms for the diabetic mothers and 143.4 gms for
the control mothers; this difference in weight gain was not
significant. Table 1 indicates the mean number of pups

per litter, mean weight of the litters, and the percentage
of males in each litter at birth for both diabetic and
control mothers. Only mothers whose offspring were
included in the study are represented.

Table 1.--Mean Litter Size, Mean Weight Per Litter, and

Mean Percent Males Per Litter From Diabetic and
Control Mothers.

 

 

Litter Wt.
Group N* Litter Size (gms.) % Males
Diabetic
Mothers 3 13.0 6.1 51.2
Control
Mothers 4 11.0 5.9 49.0

 

*N = number of mothers in each group.

Offspring

 

The mean weight just before testing at 90 days is
presented in Table 2 for male offspring in the four groups;
differences between groups were not significant. Urine
sugar, ketosis and weights were also recorded at 50 and 90
days. These data are presented in Appendix B. Offspring
of both control and diabetic mothers showed minimal levels
of sugar in the urine; positive results were not found with

Acetest on any test.

31

Table 2.--Mean Weight of Male Offspring at 90 Days.

 

 

Group Weight (gms.)
OSD* 384.2
OC* 390.0
OSDXC* 384.4
OCXSD* 387.7

 

*OSD = offspring of severely diabetic mothers; CC =
offspring of control mothers; OSDXC = offspring of severely
diabetic mothers cross fostered to control mothers; OCXSD =
offspring of control mothers cross fostered to severely
diabetic mothers.

Male Behavior Tests

 

Copulators during the weekly mating tests and on
the satiation test were determined by two criteria. Scores
from males that ejaculated on two of the three weekly
mating tests were included in the data analysis. Scores
from animals that ejaculated on the satiation test,
independent of the other tests, were included in the
satiation data. These criteria were adopted because
inclusion of data from inconsistent and non responders
skewed the scores of the group as a whole: as a result, the
data did not reflect the performance of the consistent
copulators. Table 3 presents the proportion of males in
each group who met the criterion in the weekly tests and
on the satiation test.

Means and standard errors for the behavioral

measures recorded in the three weekly tests are presented

32

Table 3.--Proportion of Copulators During Weekly Mating
Tests and Satiation Test.

 

 

Group Weekly Tests** Satiation***
OSD* 4/5 3/5

OC* 8/8 7/3
OSDXC* 8/9 8/9
OCXSD* 7/11 7/11

 

*OSD = offspring of severely diabetic mothers;
CC = offspring of control mothers; OSDXC = offspring of
severely diabetic mothers cross fostered to control mothers;
OCXSD = offspring of control mothers cross fostered to
severely diabetic mothers

**Criterion for copulators
weekly tests.

ejaculation on 2 of 3

***Criterion for copulators
satiation test only.

ejaculation on

in Table 4, scores for the satiation test are presented in
Table 5. These are measures for the first series on the
tests indicated. In addition, the mean number of ejacula-
tions on the satiation test is given for each group.

A Kruskal-Wallace test was used to determine if
there were any overall differences between groups. On
Test 1 there was an overall difference in ejaculation
latency (p<.05) and intromission frequency (p<.05). On
Test 2, the difference in ejaculation latency was signifi—
cant (p<.05); on Test 3 the difference in intromission
frequency was significant (p<.05). On the satiation test,
the difference in intromission latency (p<.05) and inter-

intromission interval (p<.05) was significant.

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35

Table 5.--Mean Behavioral Scores on Satiation Test.

 

 

OSD* OC* OSDXC* OCXSD*

ML° 60.0:54.5+ 266.7:123.0 52.6:26.5 267.4:128.3
(sec)

IL° 109.7:37.3 499.3:175.9 104.0:30.8 486.3:143.5
(sec)

EL° 390.7:72.7 1759.13818.4 424.0:45.4 lO88.7:430.0
(sec)

MF° 9.7:9.3 13.3:9.4 4.5:1.o 8.0:}.9
IF° 7.7:o.9 9.93.0 ll.8:l.l 10311.3
111° 64.1:21.6 200.2:76.3 42.3:§.5 120.4:40.4
(sec)

931° 437.3:38.0 662.3:120.7 359.8:24.9 422-9132-5
(sec)

Total E 5.3:p.3 5.4:p.4 6.6:p.5 6.1:0.8

 

*OSD = offspring of diabetic mothers; CC = offspring of
control mothers; OSDXC = offspring of severely diabetic mothers cross
fostered to control mothers; OCXSD = offspring of control mothers
cross fostered to diabetic mothers.

°ML = mount latency; IL = intromission latency; EL =
ejaculation latency; MF = mount frequency; IF = intromission frequency;

III = inter-intromission interval; PEI = post ejaculatory interval.

+Mean :_SE.

36

Due to the small number of animals in some groups,
some of the measures approached but did not reach signifi-
cance. A second analysis was done with a Mann-Whitney U
Test. First, tests were made to see if there was any
difference between offspring of severely diabetic mothers
and those cross fostered with controls; a similar analysis
was made for offSpring of control mothers and those cross
fostered with severely diabetic mothers. Only one measure
turned out to be significant from all behavior tests. On
Test 2 and 3 and on the satiation test, offspring of
severely diabetic mothers reared by their own mothers
showed fewer intromissions preceding ejaculation (p<.05)
than offspring of severely diabetic mothers who were cross
fostered. Therefore, on all measures except intromission
frequency, data from the offspring of both diabetic groups
were pooled as one group. Similarly, scores from the two
control groups were also pooled.

Table 6 shows the significance levels on the Mann-
Whitney U Test for the pooled groups. As a general trend,
latencies to the initiation of sexual behavior were

shortened in males who had diabetic mothers.

37

Table 6.--Levels of Significance on Mann-Whitney U Test
for Behavioral Measures on Weekly Mating Tests
and Satiation Test.

 

 

 

 

OSD* + OSDXC* OC* + OCXSD*
vs.
Test 1 Test 2 Test 3 Satiation

ML° p<.05 p<.05
(sec)

IL° p<.05 p<.05 p<.05
(sec)

EL° p<.05 p<.05 p<.05
(sec)

MFG

III°

(sec) p .05
PEI°

(sec)

 

*OSD = offspring of severely diabetic mothers;
OSDXC = offspring of severely diabetic mothers cross
fostered to control mothers; CC = offspring of control
mothers; OCXSD = offspring of control mothers cross
fostered to severely diabetic mothers.

°ML = mount latency; IL = intromission latency;
EL = ejaculation latency; MF = mount frequency; III =
inter-intromission interval; PEI = post ejaculatory
interval.

DISCUSSION

The sexual behavior of male offspring from diabetic
mothers was characterized by a shortened latency to the
initiation of sexual behavior. A change in temporal pacing
resulted in significantly shorter latencies to intromission
and ejaculation. Mount latency was reduced on one of the
weekly tests, and also on the satiation test. The
reduction in latencies was not attenuated by repeated
testing.

The reduction in latencies appears to result from
the prenatal diabetic environment since no difference was
found in latencies to sexual behavior between offspring of
diabetic mothers reared by their own mothers and those
cross fostered to control mothers. The only difference
attributed to cross fostering was an increase in intro-
mission frequency in cross fostered offspring of diabetic
mothers when compared to offspring of diabetics reared by
their own mothers. On all other measures, there was no
difference between pups that were reared by their own
mothers and those which were cross fostered. Although

differences in maternal behavior may have been present,

38

39

differential caregiving in general did not influence

sexual behavior.

Sexual Arousal

 

Measures of COpulatory behavior can be divided into
two categories: latency to c0pulate and rate of COpulation.
Beach (1956) has proposed that the male's sexual excite-
ment in the presence of a female increases to a point
at which he initiates c0pulation, i.e., a c0pulatory
threshold has been reached. A decrement of this threshold
is facilitated by visual and olfactory cues from the
female as well as her own solicitation behavior. The
summation of this stimulation will trigger a mount or an
intromission by the male; in the case of non copulators,
this threshold is not reached and the male habituates to
the presence of the female.

Once the c0pulatory threshold has been reached, a
secondlhypothetical mechanism, the intromission and
ejaculatory mechanism, is initiated. Stimulation provided
by copulation modifies the internal state of the animal to
bring the male to the ejaculatory threshold. The trigger
for the ejaculatory mechanism is controversial. One
possibility is that each successive intromission builds
up a level of excitement until the threshold for ejaculation
is reached. Another possibility is that the initial

intromission triggers a timing mechanism and successive

4O

intromissions maintain a constant level of input to the
timer until the culmination of the interval in ejaculation.

Male offspring of diabetic mothers in the present
study showed changes in both aspects of this dual system
of sexual motivation. The decreased intromission latency
shown by these males when compared to controls indicates
a lowering of the copulatory threshold for the initiation
of sexual behavior. The decrease in ejaculation latency
and decrease in intromission frequency in offsPring of
diabetic mothers indicates a concomitant lowering of the
ejaculatory threshold. The latency differences suggest a
difference in arousal level between males born to diabetic
mothers and males born to control mothers.

One other measure of temporal pacing, the post
ejaculatory interval, did not differ between groups. Also,
there was no difference in the number of ejaculations to
satiation between any of the groups; this would indicate
that sexual exhaustion, or lowering of sexual arousal to
the point that no intromissions occurred in a half hour
interval, followed the same total number of ejaculations
in all groups.

The performance of normal males on a sexual
exhaustion test is characterized by a U shape pattern of
response (Karen and Barfield, 1975). Facilitation of
sexual behavior, i.e., shortened ejaculation latency,
shortened inter copulatory intervals, longer durations per

intromission, and fewer intromissions to achieve ejaculation

41

characterize the intermediate stages in exhaustion tests.
Longer inter copulatory intervals and a greater number of
intromissions preceding ejaculation are seen in the first
and last series of a satiation test. In this context, male
offSpring of diabetic mothers showed an initial facilitation
compared to the behavior of control males. Additional

tests would indicate whether a U shape curve in satiation
would also be obtained for offspring of diabetic mothers,
i.e., whether the decreased latencies relative to controls
would be maintained in successive series. Although
satiation data were obtained in the present study, refusals
on the part of some females in the later series distorted
latency measures. In some instances females showed lordosis
only after persistent attempts to mount by the male. These
refusals may have been preferentially directed towards
experimental males; however, since female behavior was not
monitored throughout the tests, no conclusions can be

drawn.

Reduction in ejaculation latency, and in intro-
mission frequency, has been found in contexts other than
satiation tests. This result is found following castration
(Davidson, 1966), adrenalectomy (Bloch and Davidson, 1968),
handling of rats during c0pu1ation (Larsson, 1963),
peripheral electric shock (Barfield and Sachs, 1968), and
rewarding or aversive brain stimulation (Caggiula, 1970;
Eibergen and Caggiula, 1975). These diverse treatments

have in common their ability to increase levels of

42

behavioral arousal whether through the diffuse effects of
surgical trauma or localized stimulation in the brain.

Since peripheral stimulation is not always specific to
copulatory behavior, it probably reflects diffuse activation
of arousal mechanisms in the brain; in this study,
reactiveness to other types of stimulation was not studied
to determine if the pattern of behavior in male offspring

of diabetic mothers was specific to sexual stimulation or
whether it represented a generalized increase in responsive-

ness to external stimulation.

Adaptive Significance

 

At first glance, decrease in intromission frequency
and ejaculation latency seem to suggest a greater efficiency
in sexual behavior; the ejaculatory re5ponse can be
achieved in less time with less work. However, this
atypical patterning of sexual behavior may decrease the
reproductive efficiency of these animals. Diamond (1970)
discussed a species specific vaginal code where number and
spacing of intromissions were critical factors in initiating
neuroendocrine reflexes in the female necessary for
pregnancy to occur. Multiple intromissions result in
facilitation of Sperm transport and luteal activation;
three intromissions are sufficient for sperm tranSport
whereas more than five are necessary for luteal activation.
This corresponds to behavioral studies where only 20% of

female rats receiving three or less intromissions became

43

pregnant but 90% of those receiving approximately nine
intromissions became pregnant (Wilson, Adler and Le Boeuf,
1965; Adler, 1969; Chester and Zucker, 1970). Thus, a male
rat not conforming to the normal pattern of sexual behavior
in his species reduces his chances of reproductive success.
It is very possible that the behavior shown by offSpring

of diabetic mothers could lead to this result.

Etiology

It is difficult to assign a mechanism to the altered
pattern of sexual response seen in male offspring of
diabetic mothers, although the results do suggest an
analogy with the effects of certain stressful conditions
on temporal patterning in adult sexual behavior. Only one
study has been reported on the effects of prenatal stress
(constraint under glaring light) on sexual behavior (Ward,
1972); results indicated a decrease in ejaculations in
intact males and an increase in feminine reSponses when
given apprOpriate hormones. This was interpreted as
resulting from increased secretion of a weak androgen,
androstenedione, from the adrenal cortex as a concomitant
of stress. Consequently, the organization of sexual
behavior would be impaired paralleling the situation where
males are deprived of testosterone during develOpment.

The effects of long term stress in adult males also
results in decreased plasma testosterone (Milkovic and

Milkovic, 1966); studies have not been done on sexual

44

behavior in chronically stressed adult males but they
would be expected to show decreased ejaculatory capacity.
One possibility is that maternal diabetes in this
study functioned to produce a steady but low level of
stress in which corticosteroids were activated without also
producing large amounts of adrenal androgens. Levine and
Mullins (1966) proposed a model whereby early stress
(handling of rats in infancy) could permanently alter the
set point for adrenocortical response, resulting in a more
graded response to stressful stimuli in adulthood rather
than an all or none activation. Corticosteroids would
have to be measured in both the mother and the offSpring
at birth and maturity to determine if there was an altered
adrenal output either prenatally to alter the hypothalamo-
pituitary-adrenal system, or in adulthood to alter the

arousal value of sexual stimuli.

Conclusion

 

The effects of maternal diabetes on sexual behavior
of male offspring reflect patterns seen in conditions of
increased arousal or stress, i.e., decreased latencies to
ejaculation and decreased intromission frequency. Other
behavioral tests would be needed to determine whether this
is specific to sexual behavior or whether it represents a
phenomenon of general excitability. Studies are being
conducted presently to determine the ability of these males

to impregnate females; it is predicted that ejaculation in

45

these males will not lead to fertilization because the
intromission frequency and pacing will not function
appropriately to initiate reproductive processes in the
female. Finally, the important question of mechanisms

can only be answered by assays of hormonal changes in the
diabetic mother and her fetuses, and also by a wider range
of studies on the effects of prenatal stress on behavior,

so that meaningful comparisons can be made.

APPENDICES

APPENDIX A

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0 o e\N m.mem o o «\N N.voN o o H.36N o o H.NNH NH
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46

APPENDIX B

Table 8.--Urine Sugar and Body Weight for Male Offspring.

 

 

 

 

Day 50 Day 90
Rat
Wt. CT* TT* AT* Wt. CT TT AT
(91118) (95) (‘3) (‘35)

0513+

7 231.0 1/4 0 0 390.0 0 o 0

8 212.1 1/4 0 0 380.0 1/4 0 0

9 206.5 1/4 0 0 355.0 0 0 0
10 230.4 1/2 0 0 405.0 1/4 1/10 0
11 186.7 3/4 0 0 395.0 0 0 0
12 216.5 1/4 1/4 0 400.0 0 o 0
osnxc+

39 247.0 1/4 1/10 0 400.0 1/2 1/4 0
40 214.7 1/2 1/10 0 370.0 1/4 1/10 0
41 246.5 1/4 1/10 0 350.0 1/4 0 0
42 253.3 0 1/10 0 395.0 1/4 1/10 0
43 229.5 0 1/10 0 330.0 0 1/10 0
44 264.5 0 1/10 0 430.0 1/4 0 0
46 265.0 1/4 1/10 0 410.0 0 1/10 0
47 211.2 0 1/10 0 360.0 0 1/10 0
48 238.4 1/4 1/10 0 415.0 1/4 1/10 0
00+
19 293.5 1/4 0 0 460.0 0 0 0
20 279.8 1/4 0 0 410.0 0 1/10 0
21 229.5 1/2 1/10 0 370.0 1/4 0 0
22 254.4 0 0 0 400.0 1/4 1/10 0
24 266.3 1/4 0 0 370.0 1/4 1/10 0
56 262.0 1/4 1/10 0 360.0 0 0 0
57 255.6 1/4 1/10 0 370.0 0 0 0
58 252.4 3/4 1/10 0 405.0 1/4 1/10 0
59 222.3 3/4 1/10 0 365.0 0 0 0
ocxso+

34 242.8 1/2 1/10 0 320.0 1/2 1/10 0
35 241.2 1/4 1/10 0 310.0 1/2 1/10 0
36 227.9 1/2 0 0 410.0 1/4 0 0
37 214.9 1/2 1/10 0 350.0 0 0 0
38 223.4 0 1/10 0 350.0 1/2 1/4 0

47

48

Table 8.--Continued.

 

 

 

 

Day 50 Day 90

Rat

Wt. CT* TT* AT* Wt. CT TT AT

(W5) (58) Ha) He)
50 233.2 1/2 1/10 0 410.0 1/4 1/10 0
51 263.3 0 0 0 400.0 1/4 l/10 0
52 328.1 1/4 1/10 0 450.0 1/2 1/4 0
53 276.1 1/4 1/10 0 430.0 1/2 0 0
54 307.0 1/4 0 0 415.0 l/4 0 0
55 274.1 1/4 1/10 0 420.0 0 0 0

 

*CT = Clini—Test; TT = Tes-Tape; AT = Acetest

+ODS = offspring of severely diabetic mothers;
OSDXC = offspring of severely diabetic mothers cross
fostered to control mothers; CC = offspring of control
mothers; OCXSD = offspring of control mothers cross
fostered to severely diabetic mothers.

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