Janet Brotherton

The counting and sizing of spermatozoa from ten animal species using a coulter counter

Zählung und Größenbestimmung von Spermien zehn verschiedener Tierspezies mit einem Coulter Counter

Cortisol and transcortin in human seminal plasma and amniotic fluid as estimated by modern specific assays

Summary Cortisol concentrations in human seminal plasma, as estimated by the very specific Amersham ‘Amerlite’ luminescence immunoassay, were 176 ± 43 (85–260) nmol/l, that is, 63.7 ± 15.5 (31–94) ng/ml (mean ± SD, n = 21). This is about 60% of random levels in blood serum and is the first description of Cortisol in seminal fluid. In human amniotic fluid at 16–22 weeks of gestation, Cortisol concentrations were lower, at 72.6 ± 14.6 (63–124) nmol/l, that is, 29.3 ± 5.3 (23–45) ng/ml (n = 21). Concentrations were about 15% of random maternal serum levels in the second trimester of pregnancy. The Cortisol concentrations in both fluids were considerably higher than those reported for saliva, which has a mean of about 10 nmol/l. Transcortin (corticosteroid binding globulin, CBG), has been found in human seminal plasma and amniotic fluid for the first time. Concentrations were low, with values up to 12 μg/ml, with no significant difference between the two fluids, when using the IRE‐Megenix monoclonal iodinated radioimmunoassay. Transcortin concentrations were about 10% of levels in non‐pregnant blood serum, compared with about 0.1% for saliva. The higher concentrations of transcortin could perhaps account for the greater diffusion of Cortisol into seminal plasma and amniotic fluid. The presence of beta‐endorphin, ACTH and Cortisol in amniotic fluid, seminal fluid, ovarian follicular fluid, endometrial fluid and gastric fluid may possibly, indicate the existance of a small paracrine ACTH‐cortisol axis in the relevant secretory tissues.

The diagnostic value of plasma free testosterone in non-tumorous and tumorous hyperandrogenism *

The clinical significance of total and free testosterone (T) estimates for the diagnostic approach to hirsute patients was assessed. Plasma T was measured by radioimmunoassay and its non-protein-bound fraction was determined by equilibrium dialysis, thus facilitating the calculation of apparent free T (AFT). The cases of 162 subjects were investigated; the subjects included 75 women in whom glandular androgen release had been defined by selective catheterization. A positive linear correlation was observed between both parameters over a wide range of concentrations (T, 153 to 10,700 pg/ml; AFT, 0.8 to 342 pg/ml; P less than 0.001). Significant differences of mean T and AFT levels were found between healthy control subjects (n = 8) and subjects with non-tumorous hyperandrogenism (n = 60; P less than 0.005). Individual values overlapped considerably; elevated T (greater than 640 pg/ml; 48%) or AFT (greater than 7.2 pg/ml; 52%) were present in only half the hirsute women. However, the upper 95% confidence limits of normal for both indices were exceeded in all patients with androgen-secreting ovarian tumors (n = 7). It is concluded that the indirect estimation of AFT in addition to T is time-consuming, costly, without practical value in selecting the proper treatment, and therefore not mandatory in the routine evaluation of androgenized women.

Vasopressin: another pregnancy protein in human seminal plasma

Summary Human vasopressin (arginine‐vasopressin, AVP, antidiuretic hormone, ADH) was estimated, after protein precipitation and extraction in ethanol, using a new radioimmunoassay from Immuno Technology Service, Wijchen, Netherlands. Concentrations in human seminal plasma were 1.84 ± 1.23 (0.6–4.1) pg/ml, estimated in good duplicates in all 20 samples, where 1 pg 0.410 uIU/ml WHO 1st 77/501. This is about the same concentration as in blood serum, for which levels up to 8 pg/ml are found by the same kit. In contrast, only trace amounts of vasopressin were found in amniotic fluid at 16–22 weeks of gestation, with zero values in 8 of 19 samples, while another 9 samples showed zero in one duplicate and up to 0.46 pg/ml in the other duplicate, and one sample showed 0.09 pg/ml in good duplicates.

The interconversion of machine settings and size determinations between seven models of Coulter counter as illustrated by values for human spermatozoa

Calibration of the Coulter counter Models ZB Industrial, B Industrial D and D Industrial using 21 standard particles is reported using the same method as previously described for the Models B Medical, F and A. All the machines showed significant differences in design and sensitivity. The additional data allowed the relationship between the machine constant and the aperture diameter to be calculated and volume factors were calculated for each combination of settings on each machine. The general method of interconverting size data and machine settings between the instruments was demonstrated using human spermatozoa as an example.

The Splitting of Sperm Heads from Tails in Eight Mammalian Species and the Measurement of their Sizes

Ejaculated spermatozoa from man, the European wild boar and the bull, and spermatozoa from the cauda epididymes of the rabbit, rat, mouse, hamster and Guinea pig were treated with a sonic bath, a sonic probe, trypsin with and without prior treatment with a sulphhydryl reagent, pronase and alkalis. The fragments produced were counted and sized in an accurately calibrated Coulter Counter, Model ZB Industrial, before and after Zaponin treatment to lyse accompanying debris and the peripheral cytoplasm. Head and tail fractions were separated on sucrose gradients. Each species required different conditions for cleavage or fragmentation. Rabbit and bull spermatozoa were cleaved by the ultrasonic bath exactly into heads and tails, producing twice the number of particles with two peaks in the size distribution curves but with some 60% loss of total sperm volume which became the soluble fraction. The ultrasonic probe, and for the bull, pronase, produced the same cleavage but these more drastic treatments dissolved a considerable portion of the tail fraction. Rodent spermatozoa, especially the rat, were cleaved perfectly into heads and tails by mild trypsin treatment. All the non‐rodent spermatozoa were resistent to trypsin cleavage, although prior treatment with a sulphhydryl reagent caused swelling and subsequent trypsin action caused digestion into miscellaneous pieces. Spermatozoa from the boar and from man could not be cleaved by any of the procedures. The sonic probe produced fragmentation with progressive dissolution of the tail fragments and a single peak in the size distribution curve corresponding to small stripped heads. The soluble fraction always constituted a large proportion of the original whole spermatozoa.

Comparison of the particle size distribution curves determined manually and automatically in an accurately calibrated Coulter counter

An accurately calibrated Coulter Counter, Model ZB Industrial, was used to compare the size distribution curves of seven different standard particles obtained by calculation and after the attachment of a Channelyser, Model C-1000 coupled to an XY recorder. Comparisons were made at the modes of the curves and it was found that the automatic method consistently reported the particles to be slightly larger.